Reshaped human antibody to human interleukin-8

ABSTRACT

The present invention discloses a reshaped human antibody against human IL-8 comprising:  
     (A) L chains each comprising:  
     (1) a human L chain C region; and,  
     (2) an L chain V region comprising a human L chain FR, and an L chain CDR of mouse monoclonal antibody against human IL-8; and,  
     (B) H chains each comprising:  
     (1) a human H chain C region; and,  
     (2) an H chain V region comprising a human H chain FR, and an H chain CDR of mouse monoclonal antibody against human IL-8.  
     Since the majority of this reshaped human antibody originates in human antibody and the CDR has low antigenicity, the reshaped human antibody of the present invention has low antigenicity to humans, and can therefore be expected to be useful in medical treatment.

TECHNICAL FIELD

[0001] The present invention relates to the complementarity determiningregions (CDRs) and the variable regions (V regions) of mouse monoclonalantibody against human interleukin-8 (IL-8), to human/mouse chimericantibody against human IL-8, as well as to a reshaped human antibodywherein the complementarity determining regions of the human light chain(L chain) variable region and the human heavy chain (H chain) variableregion are substituted with the CDR of mouse monoclonal antibody againsthuman IL-8. Moreover, the present invention provides DNAs that code forthe above-mentioned antibody and its portions. The present inventionalso relates to a vector that contains the above-mentioned DNA, and moreparticularly, to an expression vector and a host transformed with saidvector. Moreover, the present invention provides a process for producingreshaped human antibody against human IL-8 as well as a process forproducing a chimeric antibody against human IL-8.

BACKGROUND ART

[0002] Interleukin-8 (IL-8) was discovered in the culture supernatant ofmonocytes stimulated with lipopolysaccharide (LPS), and is a chemokineknown also as monocyte-derived neutrophil chemotactic factor (MDNCF) orneutrophil activating protein-1 (NAP-1). IL-8 is produced by variouscells, acts on polymorphonuclear leukocytes and lymphocytes, andpossesses activity that causes chemotaxis along its concentrationgradient. In addition, not only does it induce chemotaxis inneutrophils, but it also activates neutrophilic functions such asdegranulation, the release of superoxide, and the promotion of adhesionto endothelial cells.

[0003] In inflammatory diseases, and more specifically in respiratorydiseases such as pulmonary cystic fibrosis, idiopathic pulmonaryfibrosis, adult respiratory distress syndrome, sarcoidosis and empyema,as well as in skin diseases such as psoriasis, and in chronic rheumatoidarthritis, Crohn's disease and ulcerative colitis, leukocyteinfiltration is observed pathologically at the inflamed site of thesediseases. In addition, IL-8 is detected in test samples from patientswith these diseases, suggesting that IL-8 may play a central role ininflammation. (McElvaney, N. G. et al., J. Clin. Invest., 90, 1296-1301,1992; Lynch III, J.P. et al., Am. Rev. Respir. Dis., 145, 1433-1439,1992; Donnelly, S. C. et al., Lancet, 341, 643-647, 1993; Car, B. D. etal., Am. J. Respir. Crit. Care Med., 149, 655-659, 1994; Antony, V. B.et al., J. Immunol., 151, 7216-7223, 1993; Takematsu, H. et al., Arch.Dermatol., 129, 74-80, 1993; Brennan, F. M. et al., Eur. J. Immunol.,20, 2141-2144, 1990; Izzo, R. S. et al., Scand. J. Gastroenterol., 28,296-300, 1993; Izzo, R. S. et al., Am. J. Gastroenterol., 87, 1447-1452,1992).

[0004] Subsequence to immunizing mice with human IL-8 as antigen, Ko,Y-C. et al. prepared the mouse monoclonal antibody WS-4 that binds tohuman IL-8 and inhibits the binding of human IL-8 to neutrophils as aresult of that binding, namely that neutralizes the biological activitypossessed by human IL-8. It has been clearly shown that the isotypes ofmouse monoclonal antibody WS-4 consist of a κ-type L chain and aCγl-type H chain (J. Immunol. Methods, 149; 227-235, 1992).

[0005] Known examples of antibodies against human IL-8 other than WS-4include A.5.12.14 (Boylan, A. M. et al., J. Clin. Invest., 89,1257-1267, 1992), the anti-Pep-1 antibody and anti-Pep-3 antibodydisclosed in International Patent Application No. W092-04372, and DM/C7(Mulligan, M. S. et al., J. Immunol., 150, 5585-5595, 1993).

[0006] It was also found by administration of the mouse monoclonalantibody WS-4 into experimental models using rabbits that neutrophilinfiltration is inhibited in pulmonary ischemic and reperfusion injury(Sekido, N. et al., Nature, 365, 654-657, 1993), LPS-induced dermatitis(Harada, A. et al., Internatl. Immunol., 5, 681-690, 1993) and LPS- orinterleukin-1 (IL-1)-induced arthritis (Akahoshi, T. et al., LymphokineCytokine Res., 13, 113-116, 1994).

[0007] A homologue of human IL-8 exists in rabbits, and is referred toas rabbit IL-8. Since it has been clearly shown that the mousemonoclonal antibody WS-4 cross-reacts with rabbit IL-8, and that theantibody inhibits binding of rabbit IL-8 to rabbit neutrophils (Harada,A. et al., Internatl. Immunol., 5, 681-690, 1993), these findingssuggest that anti-human IL-8 antibody would be useful as a therapeuticagent for the treatment of inflammatory diseases in humans.

[0008] Monoclonal antibodies originating in mammals other than humansexhibit a high degree of immunogenicity (also referred to asantigenicity) in humans. For this reason, even if mouse antibody isadministered to humans, as a result of its being metabolized as aforeign substance, the half life of mouse antibody in humans isrelatively short, thus preventing its anticipated effects from beingadequately demonstrated. Moreover, human anti-mouse antibody that isproduced in response to administered mouse antibody causes an immuneresponse that is both uncomfortable and dangerous for the patient,examples of which include serum sickness or other allergic response. Forthis reason, mouse antibody cannot be administered frequently to humans.

[0009] In order to resolve these problems, a process for producing ahumanized antibody was developed. Mouse antibody can be humanized by twomethods. The simpler method involves producing a chimeric antibody inwhich the variable region (V region) is derived from the original mousemonoclonal antibody, and the constant region (C region) is derived froma suitable human antibody. Since the resulting chimeric antibodycontains the variable region of the mouse antibody in its complete form,it has identical specificity to the original mouse antibody, and can beexpected to bind to antigen.

[0010] Moreover, in the chimeric antibody, since the proportion ofprotein sequences derived from an animal other than human issubstantially reduced in comparison to the original mouse antibody, itis predicted to have less immunogenicity in comparison to the originalmouse antibody. Although the chimeric antibody binds well to antigen andhas low immunogenicity, there is still the possibility of an immuneresponse to the mouse variable region occurring, however (LoBuglio, A.F.et al., Proc. Natl. Acad. Sci. USA, 86, 4220-4224, 1989).

[0011] Although the second method for humanizing mouse antibody is morecomplexed, the latent immunogenicity of the mouse antibody is reducedconsiderably. In this method, only the complementarity determiningregion (CDR) is grafted from the variable region of mouse antibody ontothe human variable region to create a reshaped human variable region.However, in order to approximate more closely the structure of the CDRof the reshaped human variable region to the structure of the originalmouse antibody, there are cases in which it may be necessary to graft aportion of the protein sequence of the framework region (FR) supportingthe CDR from the variable region of the mouse antibody to the humanvariable region.

[0012] Next, these reshaped human variable regions are linked to thehuman constant region. Those portions derived from non-human proteinsequences consist only of the CDR and a very slight portion of the FR inthe humanized antibody. CDR is composed of hyper-variable proteinsequences, and these do not exhibit species specificity. For thisreason, the reshaped human antibody that contains the mouse CDRs oughtnot to have immunogenicity stronger than that of a natural humanantibody containing human CDRs.

[0013] Additional details regarding reshaped human antibodies can befound by referring to Riechmann, L. et al., Nature, 332, 323-327, 1988;Verhoeyen, M. et al., Science, 239, 1534-1536, 1988; Kettleborough, C.A. et al., Protein Eng., 4, 773-783, 1991; Maeda, H. et al., Hum.Antibodies Hybridomas, 2, 124-134, 1991; Gorman, S. D. et al., Proc.Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest, P. R. et al.,Bio/Technology, 9, 266-271, 1991; Co, M. S. et al., Proc. Natl. Acad.Sci. USA, 88, 2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad. Sci.USA, 89, 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148, 1149-1154,1992; and, Sato, K. et al., Cancer Res., 53, 851-856, 1993.

DISCLOSURE OF THE INVENTION

[0014] As stated above, although reshaped human antibodies are predictedto be useful for the purpose of therapy, there are no known reshapedhuman antibodies against human IL-8. Moreover, there are no standardprocesses that can be applied universally to an arbitrary antibody. forproducing reshaped human antibody. Thus, various contrivances arenecessary to create a reshaped human antibody that exhibits sufficientbinding activity and/or neutralizing activity with respect to a specificantigen (for example, Sato, K. et al., Cancer Res., 53, 851-856, 1993).The present invention provides an antibody against human IL-8 having alow degree of immunogenicity.

[0015] The present invention provides a reshaped human antibody againsthuman IL-8. The present invention also provides a human/mouse chimericantibody that is useful in the production process of said reshaped humanantibody. Moreover, the present invention also provides a fragment ofreshaped human antibody. In addition, the present invention provides anexpression system for producing chimeric antibody and reshaped humanantibody and fragments thereof. Moreover, the present invention alsoprovides a process for producing chimeric antibody against human IL-8and fragments thereof, as well as a process for producing reshaped humanantibody against human IL-8 and fragments thereof.

[0016] More specifically, the present invention provides:

[0017] (1) an L chain V region of mouse monoclonal antibody againsthuman IL-8; and,

[0018] (2) an H chain V region of mouse monoclonal antibody againsthuman IL-8.

[0019] Moreover, the present invention provides:

[0020] (1) an L chain comprising a human L chain C region, and an Lchain V region of mouse monoclonal antibody against human IL-8; and,

[0021] (2) an H chain comprising a human H chain C region, and an Hchain V region of mouse monoclonal antibody against human IL-8.

[0022] Moreover, the present invention also provides chimeric antibodyagainst human IL-8 comprising:

[0023] (1) L chains each comprising a human L chain C region, and an Lchain V region of mouse monoclonal antibody against human IL-8; and,

[0024] (2) H chains each comprising a human H chain C region, and an Hchain V region of mouse monoclonal antibody against human IL-8.

[0025] Moreover, the present invention provides:

[0026] (1) an L chain V region of mouse monoclonal antibody WS-4 againsthuman IL-8; and,

[0027] (2) an H chain V region of mouse monoclonal antibody WS-4 againsthuman IL-8.

[0028] Moreover, the present invention also provides:

[0029] (1) an L chain comprising a human L chain C region, and an Lchain V region of mouse monoclonal antibody WS-4 against human IL-8;and,

[0030] (2) an H chain comprising a human H chain C region, and an Hchain V region of mouse monoclonal antibody WS-4 against human IL-8.

[0031] In addition, the present invention provides chimeric antibodyagainst human IL-8 comprising:

[0032] (1) L chains each comprising a human L chain C region, and an Lchain V region of mouse monoclonal antibody WS-4 against human IL-8;and,

[0033] (2) H chains each comprising a human H chain C region, and an Hchain V region of mouse monoclonal antibody WS-4 against human IL-8.

[0034] Moreover, the present invention provides:

[0035] (1) CDR of an L chain V region of monoclonal antibody againsthuman IL-8; and,

[0036] (2) CDR of an H chain V region of monoclonal antibody againsthuman IL-8.

[0037] Moreover, the present invention also provides:

[0038] (1) CDR of an L chain V region of mouse monoclonal antibodyagainst human IL-8; and,

[0039] (2) CDR of an H chain V region of mouse monoclonal antibodyagainst human IL-8.

[0040] Moreover, the present invention also provides a reshaped human Lchain V region of an antibody against human IL-8 comprising:

[0041] (1) framework regions (FRs) of a human L chain V region; and,

[0042] (2) CDRs of an L chain V region of mouse monoclonal antibodyagainst human IL-8;

[0043] as well as a reshaped human H chain V region of antibody againsthuman IL-8 comprising:

[0044] (1) FRs of a human H chain V region; and,

[0045] (2) CDRs of an H chain V region of mouse monoclonal antibodyagainst human IL-8.

[0046] Moreover, the present invention provides a reshaped human L chainof antibody against human IL-8 comprising:

[0047] (1) a human L chain C region; and,

[0048] (2) an L chain V region comprising human L chain FRs and L chainCDRs of mouse monoclonal antibody against human IL-8;

[0049] as well as a reshaped human H chain of antibody against humanIL-8 comprising:

[0050] (1) a human H chain C region; and,

[0051] (2) an H chain V region comprising human H chain FRs and H chainCDRs of mouse monoclonal antibody against human IL-8.

[0052] In addition, the present invention also provides reshaped humanantibody against human IL-8 comprising:

[0053] (A) L chains each comprising:

[0054] (1) a human L chain C region; and,

[0055] (2) an L chain V region comprising FRs of a human L chain, andCDRs of an L chain of mouse monoclonal antibody against human IL-8; aswell as

[0056] (B) H chains each comprising:

[0057] (1) a human H chain C region; and,

[0058] (2) an H chain V region comprising FRs of a human H chain, andCDRs of an H chain of mouse monoclonal antibody against human IL-8.

[0059] More specifically, the present invention provides:

[0060] (1) CDRs of an L chain V region of mouse monoclonal antibody WS-4against human IL-8 having the following sequences or a portion thereof:

[0061] CDR1: Arg Ala Ser Glu Ile Ile Tyr Ser Tyr Leu Ala

[0062] CDR2: Asn Ala Lys Thr Leu Ala Asp

[0063] CDR3: Gln His His Phe Gly Phe Pro Arg Thr as well as

[0064] (2) CDRs of an H chain V region of mouse monoclonal antibody WS-4against human IL-8 having the following sequences or a portion thereof:

[0065] CDR1: Asp Tyr Tyr Leu Ser

[0066] CDR2: Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser AlaSer Val Lys Gly

[0067] CDR3: Glu Asn Tyr Arg Tyr Asp Val Glu Leu Ala Tyr

[0068] Moreover, the present invention provides a reshaped human L chainV region of antibody against human IL-8 comprising:

[0069] (1) framework regions (FRs) of a human L chain V region; and,

[0070] (2) CDRs of an L chain V region of mouse monoclonal antibody WS-4against human IL-8; as well as

[0071] a reshaped human H chain V region of antibody against human IL-8comprising:

[0072] (1) FRs of a human H chain V region; and,

[0073] (2) CDRs of an H chain V region of monoclonal antibody WS-4against human IL-8.

[0074] Moreover, the present invention provides a reshaped human L chainof antibody against human IL-8 comprising:

[0075] (1) a human L chain C region; and,

[0076] (2) an L chain V region comprising FRs of a human L chain, andCDRs of an L chain of mouse monoclonal antibody WS-4 against human IL-8;as well as

[0077] a reshaped human H chain of antibody against human IL-8comprising:

[0078] (1) a human H chain C region; and,

[0079] (2) an H chain V region comprising FRs of a human H chain, andCDRs of an H chain of monoclonal antibody WS-4 against human IL-8.

[0080] In addition, the present invention also provides a reshaped humanantibody against human IL-8 comprising:

[0081] (A) L chains each comprising:

[0082] (1) a human L chain C region; and,

[0083] (2) an L chain V region comprising FRs of a human L chain andCDRs of an L chain of mouse monoclonal antibody WS-4 against human IL-8;and

[0084] (B) H chains each comprising:

[0085] (1) a human H chain C region; and,

[0086] (2) an H chain V region comprising FRs of a human H chain andCDRs of an H chain of mouse monoclonal antibody WS-4 against human IL-8.

[0087] Examples of the above-mentioned FRs of a human L chain includethose having the following amino acid sequences or a portion thereof:

[0088] FR1: Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly Asp Arg Val Thr Ile Thr Cys

[0089] FR2: Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

[0090] FR3: Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys

[0091] FR4: Phe Gly Gln Gly Thr Lys Val Glu Ile Lys or,

[0092] FR1: Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly Asp Arg Val Thr Ile Thr Cys

[0093] FR2: Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

[0094] FR3: Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp TyrThr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys

[0095] FR4: Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

[0096] Examples of the above-mentioned FRs of a human H chain includethose having the following amino acid sequences or a portion thereof:

[0097] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0098] FR2: Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Leu Val Gly

[0099] FR3: Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0100] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser;

[0101] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0102] FR2: Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Trp Val Gly

[0103] FR3: Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0104] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser;

[0105] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0106] FR2: Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val Gly

[0107] FR3: Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0108] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser;

[0109] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0110] FR2: Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly

[0111] FR3: Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0112] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser;

[0113] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0114] FR2: Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val Gly

[0115] FR3: Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0116] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser;

[0117] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0118] FR2: Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Val Gly

[0119] FR3:Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0120] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser;

[0121] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0122] FR2: Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly

[0123] FR3: Arg Phe Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0124] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser; or,

[0125] FR1: Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

[0126] FR2: Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Trp Val Gly

[0127] FR3: Arg Phe Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg

[0128] FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[0129] In addition, the present invention also relates to DNA that codesfor polypeptide that comprises the above-mentioned various antibodies,and their fragments. The present invention also relates to a vector thatcontains the above-mentioned DNA, an example of which is an expressionvector. Moreover, the present invention provides a host that istransformed by the above-mentioned vector.

[0130] Moreover, the present invention also provides a process forproducing chimeric antibody against human IL-8, and its fragments, aswell as a process for producing reshaped human antibody against humanIL-8, and its fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0131]FIG. 1 indicates the expression vectors HEF-VL-gκ and HEF-VH-gγl,containing the human elongation factor-1α (HEF-1α) promoter/enhancer,which are useful for expression of the L chain and H chain,respectively, of the antibody of the present invention.

[0132]FIG. 2 is a graph indicating the results of ELISA for confirmationof the binding ability to human IL-8 of the chimeric WS-4 antibody(chL/chH) of the present invention secreted into the culture medium ofCOS cells.

[0133]FIG. 3 is a diagram of the construction of DNA that codes for theamino acid sequences of each of the first version “a” (RVHa) of the Hchain V region of reshaped human WS-4 antibody of the present invention(A), and the first version “a” (RVLa) of the L chain V region ofreshaped human WS-4 antibody (B).

[0134]FIG. 4 is a graph indicating the results of ELISA for comparingthe binding ability to human IL-8 of the L chain V region (RVLa) and theH chain V region (RVHa) of the reshaped human WS-4 antibody of thepresent invention in combination with, respectively, the H chain Vregion of chimeric WS-4 antibody (chH) and the L chain V region ofchimeric WS-4 antibody (chL) expressed in COS cells, with that of thechimeric WS-4 antibody (chL/chH) of the present invention secreted intothe culture medium of COS cells.

[0135]FIG. 5 is a graph indicating the results of ELISA for comparingthe binding ability against human IL-8 of 8 types of reshaped human WS-4antibody containing the RVLa of the present invention (RVLa/RVHa,RVLa/RVHb, RVLa/RVHc, RVLa/RVHd, RVLa/RVHe, RVLa/RVHf, RVLa/RVHg andRVLa/RVHh) secreted into the culture medium of COS cells, with that ofthe chimeric WS-4 antibody (chL/chH) of the present invention secretedinto the culture medium of COS cells.

[0136]FIG. 6 is a graph indicating the results of ELISA for comparingthe binding ability to human IL-8 of 8 types of reshaped human WS-4antibody containing the second version RVLb of the present invention(RVLb/RVHa, RVLb/RVHb, RVLb/RVHc, RVLb/RVHd, RVLb/RVHe, RVLb/RVHf,RVLb/RVHg and RVLb/RVHh) produced in the culture supernatant of COScells, with that of the chimeric WS-4 antibody (chL/chH) of the presentinvention secreted into the culture medium of COS cells.

[0137]FIG. 7 is a graph indicating the results of ELISA for comparingthe binding abilities to human IL-8 of the purified reshaped human WS-4antibodies RVLa/RVHg and RVLb/RVHg of the present invention and thepurified chimeric WS-4 antibody (chL/chH) of the present invention.

[0138]FIG. 8 is a graph indicating the results of ligand receptorbinding inhibition assays for comparison of the ability to inhibitbinding of IL-8 to the IL-8 receptor, of the purified reshaped humanantibodies RVLa/RVHg and RVLb/RVHg of the present invention, with thatof the mouse WS-4 antibody and the chimeric WS-4 antibody (chL/chH) ofthe present invention.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

[0139] Cloning of DNA Coding for Mouse V Region

[0140] In order to clone a gene that codes for the V region of mousemonoclonal antibody against human IL-8, it is necessary to prepare ahybridoma that produces mouse monoclonal antibody against human IL-8 forthe acquisition of such a gene. After the extraction of mRNA from thehybridoma, the mRNA is converted into single-stranded cDNA according toknown methods, followed by amplification of the target DNA using thepolymerase chain reaction (PCR) to obtain the gene. An example of asource of this gene is the hybridoma WS-4, which produces mousemonoclonal antibody against human IL-8, produced by Ko, Y.C. et al. Theprocess for preparing this hybridoma is described in J. Immunol.Methods, 149, 227-235, 1992, and is described later as Reference Example1.

[0141] (1) Extraction of Total RNA

[0142] In order to clone the target DNA that codes for the V region ofmouse monoclonal antibody against human IL-8, total RNA can be obtainedby disrupting the hybridoma cells by guanidine thiocyanate treatment andperforming cesium chloride density gradient centrifugation (Chirgwin,J.M. et al., Biochemistry, 18, 5294-5299, 1979). Furthermore, othermethods that are used during the cloning of genes, such as that in whichdetergent treatment and phenol treatment are performed in the presenceof a ribonuclease (RNase) inhibitor such as vanadium complex (Berger,S.L. et al., Biochemistry, 18, 5143-5149, 1979), can also be used.

[0143] (2) cDNA Synthesis

[0144] Next, single-stranded cDNA complementary to mRNA can be obtainedby treating the total RNA with reverse transcriptase using oligo(dT), anoligonucleotide complementary to the poly (A) tail located at the 3′ endof mRNA, as primer, and the mRNA contained in the total RNA obtained inthe above manner as template (Larrick, J. W. et al., Bio/Technology, 7,934-938, 1989). In addition, a random primer may also be used at thesame time. Furthermore, in the case that it is desired first to isolatemRNA, this may be done by applying the total RNA to a column ofoligo(dT)-cellulose, to which the poly(A) tail of mRNA binds.

[0145] (3) Amplification of DNA Coding for V Region by Polymerase ChainReaction

[0146] Next, cDNA that codes for the above-mentioned V region isspecifically amplified using the polymerase chain reaction (PCR). Inorder to amplify the kappa (κ) type L chain V region of mouse monoclonalantibody, the 11 types of oligonucleotide primers shown in SEQ ID Nos: 1to 11 (Mouse Kappa Variable; MKV) and the oligonucleotide primer shownin SEQ ID No: 12 (Mouse Kappa Constant; MKC) are used as the 5′ terminalprimer and the 3′ terminal primer, respectively. The above-mentioned MKVprimers hybridize to the DNA sequence that codes for the mousekappa-type L chain leader sequence, while the above-mentioned MKC primerhybridizes to the DNA sequence that codes for the mouse kappa-type Lchain C region.

[0147] In order to amplify the H chain V region of mouse monoclonalantibody, the 12 types of oligonucleotide primers shown in SEQ ID Nos:13 to 24 (Mouse Heavy Variable; MHV) and the oligonucleotide primershown in SEQ ID No: 25 (Mouse Heavy Constant; MHC) are used as the 5′terminal primer and the 3′ terminal primer, respectively. Theabove-mentioned MHV primers hybridize to the DNA sequence that codes forthe mouse H chain leader sequence, while the above-mentioned MHC primerhybridizes to the DNA sequence that codes for the mouse H chain Cregion.

[0148] Furthermore, all 5′ terminal primers (MKV and MHV) contain thesequence GTCGAC that provides a SalI restriction enzyme cleavage sitenear the 3′ terminus, while both 3′-terminal primers (MKC and MHC)contain the nucleotide sequence CCCGGG that provides an XmaI restrictionenzyme cleavage site near the 5′ terminus. These restriction enzymecleavage sites are used for the subcloning of target DNA fragments thatcode for both V regions into the respective cloning vectors. In the casethat these restriction enzyme cleavage sites are also present in thetarget DNA sequence that codes for both V regions, other restrictionenzyme cleavage sites should be used for subcloning into the respectivecloning vectors.

[0149] (4) Isolation of DNA Coding for V Region

[0150] Next, in order to obtain the DNA fragment that codes for thetarget V region of mouse monoclonal antibody, the PCR amplificationproducts are separated and purified on a low melting-point agarose gelor by a column [PCR Product Purification kit (QIAGEN PCR PurificationSpin Kit: QIAGEN); DNA purification kit (GENECLEAN II, BIO101). A DNAfragment is obtained that codes for the target V region of mousemonoclonal antibody by enzyme treatment of the purified amplificationproduct with the restriction enzymes SalI and XmaI.

[0151] Further, by cleaving a suitable cloning vector, like plasmidpUC19, with the same restriction enzymes, SalI and XmaI, andenzymatically linking the above-mentioned DNA fragment to this pUC19, aplasmid is obtained which contains a DNA fragment that codes for thetarget V region of mouse monoclonal antibody. Determination of thesequence of the cloned DNA can be performed in accordance with anyroutine method, an example of which is the use of an automated DNAsequencer (Applied Biosystems). Cloning and sequence determination ofthe target DNA are described in detail in Examples 1 and 2.

[0152] Complementarity Determining Regions (CDRs)

[0153] The present invention also provides hyper-V region orcomplementarity determining region (CDR) of the V region of mousemonoclonal antibody against human IL-8. V regions of both the L chainand H chain of the antibody form an antigen binding site. These regionson the L chain and the H chain have a similar basic structure. The Vregions of both chains contain four framework regions for which thesequence is relatively conserved, and these four framework regions arelinked by three hyper-V regions or CDR (Kabat, E.A. et al, “Sequences ofProteins of Immunological Interest”, US Dept. Health and Human Services,1991).

[0154] The majority of the portions of the above-mentioned fourframework regions (FR) have a β-sheet structure, and the three CDRs formloops. The CDRs may form a portion of the β sheet structure in somecases. The three CDRs are maintained at extremely close positionsthree-dimensionally by the FRs, and contribute to formation of theantigen binding site together with three paired CDRs. The presentinvention provides CDRs that are useful as components of humanizedantibody, as well as the DNA that codes for them. These CDRs can bedetermined from the experimental rules of Kabat, E.A. et al. “Sequencesof Proteins of Immunological Interest”, by comparing V region sequenceswith known amino acid sequences of the V region, a detailed explanationof which is provided in Embodiment 3.

[0155] Preparation of Chimeric Antibody

[0156] Prior to designing a reshaped human V region of antibody againsthuman IL-8, it is necessary to confirm whether the CDRs used actuallyform an antigen-binding region. Chimeric antibody was prepared for thispurpose. In order to prepare chimeric antibody, it is necessary toconstruct DNA that codes for the L chain and the H chain of chimericantibody. The basic method for constructing both DNA involves linkingthe respective DNA sequences of the mouse leader sequence observed inPCR-cloned DNA and the mouse V region sequence to a DNA sequence thatcodes for human C region already present in a mammalian cell expressionvector.

[0157] The above-mentioned human antibody C regions can be any human Lchain C region and any human H chain C region, and with respect to the Lchain, examples include human L chain Cκ or Cλ, while with respect tothe H chain if IgG, examples include Cγ1, Cγ2, Cγ3 or Cγ4 (Ellison, J.et al., DNA, 1, 11-18 (1981), Takahashi, N. et al., Cell, 29, 671-679(1982), Krawinkel, U. et al., EMBO J., 1, 403-407 (1982)), or otherisotypes.

[0158] Two types of expression vectors are prepared for production ofchimeric antibody, namely, an expression vector that contains DNA thatcodes for mouse L chain V region and human L chain C region under thecontrol of an enhancer/promoter expression control region, and anexpression vector that contains DNA that codes for mouse H chain Vregion and human H chain C region under the control of anenhancer/promoter type of expression control region. Next, host cellssuch as mammalian cells are simultaneously transformed by both of theseexpression vectors, and the transformed cells are cultured either invitro or in vivo to produce chimeric antigen (e.g. W091-16928).

[0159] Alternatively, DNA that codes for mouse L chain V region andhuman L chain C region and DNA that codes for mouse H chain V region andhuman H chain C region can be introduced into a single expressionvector, host cells are transformed using said vector, and are thencultured either in vitro or in vivo to produce chimeric antibody.

[0160] The production of chimeric antibody from monoclonal antibody WS-4is described in Embodiment 4.

[0161] cDNA that codes for mouse WS-4 κ-type L chain leader sequence andthe V region is cloned using PCR, and linked to an expression vectorthat contains human genome DNA that codes for the human L chain Cκregion. Similarly, cDNA that codes for the H chain leader sequence and Vregion of mouse WS-4 antibody is cloned using PCR and linked to anexpression vector that contains human genome DNA that codes for humanCγ1 region.

[0162] More specifically, suitable nucleotide sequences are introducedat the 5′ and 3′ termini of cDNAs that code for the V regions of mouseWS-4 antibody using specially designed PCR primers so that (1) they canbe easily inserted into the expression vector, and (2) they functionsuitably in said expression vector (for example, transcriptionefficiency is improved by introducing a Kozak sequence in the presentinvention).

[0163] Next, DNA that codes for the V region of mouse WS-4 antibodyobtained by amplification by PCR using these primers is introduced intoHEF expression vector (see FIG. 1) that already contains the desiredhuman C region. These vectors are suitable for transient or stableexpression of antibody genetically engineered in various mammalian cellsystems.

[0164] When the antigen-binding activity of the chimeric WS-4 antibodyprepared in this manner was tested, the chimeric WS-4 antibodydemonstrated binding activity to human IL-8 (see FIG. 2). Thus, it wasconcluded that the correct mouse V region had been cloned, and thecorrect sequence had been determined.

[0165] Design of Reshaped Human WS-4 Antibody

[0166] In order to prepare a reshaped human antibody in which the CDRsof mouse monoclonal antibody are grafted onto human antibody, it isdesirable that there be a high degree of homology between the amino acidsequences of the FRs of the mouse monoclonal antibody having the CDRs tobe grafted, and the amino acid sequences of the FRs of the humanmonoclonal antibody into which the CDRs are to be grafted.

[0167] For this purpose, the human V regions to serve as the basis fordesigning the V regions of the reshaped human WS-4 antibody can beselected by comparing the amino acid sequences of the FRs of the mousemonoclonal antibody with the amino acid sequence of the FR of the humanantibodies. More specifically, the V regions of the L and H chains ofmouse WS-4 antibody were compared with all known human V regions foundin the database of the National Biomedical Research Foundation (NBRF)using the genetic analytical software, GENETEX (Software DevelopmentCo., Ltd.).

[0168] In a comparison with known human L chain V regions, the L chain Vregion of mouse WS-4 antibody was found to resemble most closely that ofhuman antibody HAU (Watanabe, S. et al., Hoppe-Seyler's Z. Physiol.Chem., 351, 1291-1295, 1970), having homology of 69.2%. On the otherhand, in a comparison with known human antibody H chain V regions, the Hchain V region of WS-4 antibody was found to resemble most closely thatof human antibody VDH26 (Buluwela, L. et al., EMBO J., 7, 2003-2010,1988), having homology of 71.4%.

[0169] In general, homology of the amino acid sequences of mouse Vregions to the amino acid sequences of human V regions is less than thehomology to amino acid sequences of mouse V regions. This indicates thatthe V region of mouse WS-4 antibody does not completely resemble thehuman V region, and at the same time, indicates that humanization ofmouse WS-4 V region is the best way to solve the problem ofimmunogenicity in human patients.

[0170] The V region of mouse WS-4 antibody was further compared with theconsensus sequence of human V region subgroup defined by Kabat, E. A. etal., (1991), Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office, to compare between V region FR. Those results are shownin Table 1. TABLE 1 Homology (%) Between FR of Mouse WS-4 V Region andFR of the Consensus Sequence of the Human V Regions of Various SubgroupsHSGI HSGII HSGIII HSGIV A. FR in L Chain V Region 64.4 51.3 57.3 57.5 B.FR in H Chain V Region 46.9 40.9 62.3

[0171] The FRs of the L chain V region of mouse WS-4 antibody mostclosely resembled the consensus sequence of FR of the human L chain Vregion subgroup I (HSGI), having homology of 64.4%. On the other hand,the FRs of the H chain V region of mouse WS-4 antibody most closelyresembled the consensus sequence of human H chain V region subgroup III(HSGIII), having homology of 62.3%.

[0172] These results support the results obtained from the comparisonwith known human antibodies, the L chain V region of human antibody HAUbelonging to human L chain V region subgroup I, and the H chain V regionof human antibody VDH26 belonging to human H chain V region subgroupIII. In order to design the L chain V region of reshaped human WS-4antibody, it is probably best to use a human L chain V region belongingto subgroup I (HSGI), while in order to design the H chain V region ofreshaped human WS-4 antibody,. it is probably best to use the H chain Vregion of a human antibody belonging to subgroup III (HSGIII).

[0173] In a comparison with the L chain V region of known humanantibodies, the L chain V region of mouse antibody WS-4 most closelyresembled the L chain V region of human antibody REI, a member ofsubgroup I of human L chain V region. Thus, the FR of REI were used indesigning the L chain V region of reshaped human WS-4 antibody. Withinthese human FR based on REI, there are differences in five amino acids(at positions 39, 71, 104, 105 and 107; see Table 2) in comparison withthe human REI documented in the original literature (Palm, W. et al.,Hoppe-Seyler's Z. Physiol. Chem., 356, 167-191, 1975; and, Epp, 0. etal., Biochemistry, 14, 4943-4952, 1975).

[0174] The amino acid numbers shown in the table are based on theexperience of Kabat, E. A. et al. (1991). The changes in the two aminoacids at positions 39 and 71 were same changes caused by the amino acidspresent in the FR of the L chain V region of rat CAMPATH-1H antibody(Riechmann, et al., 1988). According to Kabat, et al. (1991), thechanges in the other three amino acids in FR4 (positions 104, 105 and107) are based on the J region from other human κL chains, and do notdeviate from humans.

[0175] Two versions of the L chain V region of reshaped human WS-4antibody were designed. In the first version RVLa, FR was identical tothe FR based on REI present in reshaped human CAMPATH-1H antibody(Riechmann, et al., 1988), while the CDR was identical to the CDR in theL chain V region of mouse WS-4 antibody. The second version, RVLb, wasbased on RVLa, and differed only by one amino acid at position 71 inhuman FR3. As defined by Chothia, C. et al., J. Mol. Biol., 196,901-917, 1987, residue 71 is a portion of the canonical structure of theCDR1 of the L chain V region.

[0176] Amino acid at this position is predicted to directly affect thestructure of the CDR1 loop of the L chain V region, and for this reason,it considered to have a significant effect on antigen binding. In RVLbof the L chain V region of reshaped human WS-4 antibody, thephenylalanine at position 71 is changed to tyrosine. Table 2 shows therespective amino acid sequences of the L chain V region of mouse WS-4antibody, the FR of the modified REI for use in reshaped humanCAMPATH-1H antibody (Riechmann, et al., 1988) and the two versions ofthe L chain V region of reshaped human WS-4 antibody. TABLE 2 Design ofL Chain V Region of Reshaped Human WS-4 1   2 3 412345678901234567890123 45678901234 567890123456789 WS-4LDIQMTQSPASLSASVGETVTITC RASEIIYSYLA WYQQKQGKSPQLLVY REIDIQMTQSPSSLSASVGDRVTITC WYQQKPGKAPKLLIY RVLa DIQMTQSPSSLSASVGDRVTITCRASEIIYSYLA WYQQKPGKAPKLLIY RVLb ------------------------ -------------------------- FR1 CDR1 FR2 5 6  7  8 9 012345678901234567890123456789012345678 901234567 WS-4L NAKTLADGVSSRFSGSGSGTQFSLRISSLQPEDFGSYYC QHHFGFPRT REIGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC RVLa NAKTLADGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QHHFGFPRT RVLb ---------------------Y----------------- --------- CDR2 FR3 CDR3 10 8901234567WS-4L FGGGTKLELK REI FGQGTKVEIK RVLa FGQGTKVEIK RVLb ---------- FR4

[0177] The FR in the H chain V region of mouse WS-4 antibody mostclosely resemble the human H chain V region belonging to subgroup III(Table 1).

[0178] In a comparison with known human H chain V regions, the H chain Vregion of mouse WS-4 antibody most closely resembled the H chain Vregion of human antibody VDH26, a member of subgroup III of the human Hchain V region, from FR1 to FR3 (Buluwela, L. et al., EMBO J., 7,2003-2010, 1988). With respect to FR4, since the FR4 sequence of VDH26was not reported, it was decided to use the amino acid sequence of FR4of human antibody 4B4 belonging to subgroup III (Sanz, I. et al., J.Immunol., 142, 883-887, 1989). These human H chain V regions were usedas the basis for designing the H chain V region of reshaped human WS-4antibody.

[0179] Eight versions of the H chain V region of reshaped human WS-4antibody were designed. In all eight versions, human FR1, FR2 and FR3were based on FR1, FR2 and FR3 of human antibody VDH26, while FR4 wasbased on FR4 of human antibody 4B4. Mouse CDR was identical to the CDRof the H chain V region of mouse WS-4 antibody.

[0180] Tables 3 and 4 show the respective amino acid sequences of the Hchain V region of mouse WS-4 antibody, the template FR1 through FR3 ofhuman antibody VDH26, FR4 of human antibody 4B4, and the 8 versions ofthe H chain V region of reshaped human WS-4 antibody. TABLE 3 Design ofH Chain V Region of Reshaped Human WS-4 Antibody (Followed by Table 4)1  2  3 123456789012345678901234567890 12345 WS-4HEVKLVESGGGLIQPGDSLRLSCVTSGFTFS DYYLS VDH26EVQLLESGGGLVQPGGSLRLSCAASGFTFS RVHa ˜ h EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYLS FR1 CDR1 4 5  6 67890123456789 012ABC3456789012345 WS-4HWVRQPPGKALEWVG LIRNKANGYTREYSASVKG VDH26 WVRQAQGKGLELVG RVHaWVRQAQGKGLELVG LIRNKANGYTREYSASVKG RVHb -----------W--------------------- RVHc -----P-------- ------------------- RVHd-----P-----W-- ------------------- RVHe ----PP-----W--------------------- RVHf -----P--A--W-- ------------------- RVHg-----P-----W-- ------------------- RVHh -----------W--------------------- FR2 CDR2

[0181] TABLE 4 Design of H Chain V Region of Reshaped Human WS-4(Following on Table 3) 7  8  9 10 67890123456789012ABC3456789)1234567890A8C12 WS-4H RFTISRDDSQSILYLQMNTLRGEDSATYYCAR ENYRYDVELAY VDH26RLTISREDSKNTLYLQMSSLKTEDLAVYYCAR RVHa RLTISREDSKNTLYLQMSSLKTEDLAVYYCARENYRYDVELAY RVHb -------------------------------- ----------- RVHc-------------------------------- ----------- RVHd-------------------------------- ----------- RVHe-------------------------------- ----------- RVHf-------------------------------- ----------- RVHg-F------------------------------ ----------- RVHh-F------------------------------ ----------- FR3 CDR3 11 34567890123WS-4H WGQGTLVTVSA 4B4 WGQGTLVTVSS RVHh WGQGTLVTVSS FP4

[0182] Preparation of DNA Coding for V Region of Reshaped Human WS-4Antibody

[0183] Preparation of the V region of reshaped human WS-4 antibody isdescribed in detail in Example 5.

[0184] DNAs that code for the respective first versions of the L chainand H chain V regions of reshaped human WS-4 antibody were synthesized.It was then confirmed that the entire DNA sequence of version “a” of theL chain and H chain V regions of reshaped human WS-4 antibody codes forthe correct amino acid sequence by sequence determination. The sequenceof version “a” of the L chain V region of reshaped human WS-4 antibodyis shown in SEQ ID NO: 62, while the sequence of version “a” of the Hchain V region of reshaped human WS-4 antibody is shown in SEQ ID NO:38.

[0185] DNAs that code for other versions of V region of reshaped humanWS-4 antibody were prepared using a slight variation of the publiclydisclosed PCR-mutation induction method (Kammann, M. et al., NucleicAcids Res., 17, 5404, 1989) with the first version “a” as the template.As previously described in relation to the design of the V region of thereshaped human WS-4 antibody, DNA that codes for one additional versionof the L chain V region of reshaped human WS-4 antibody (version “b”),as well as DNA that code for seven additional versions of the H chain Vregion of reshaped human WS-4 antibody (versions “b”, “c”, “d”, “e”,“f”, “g” and “h”) were prepared.

[0186] These additional versions contained slight changes in a series ofamino acid sequences from the first version, and these changes in theamino acid sequences were achieved by making slight changes in the DNAsequence using PCR mutation induction. A PCR primer was designed thatintroduces the required change in the DNA sequence. After a series ofPCR reactions, the PCR product was cloned followed by sequencedetermination to confirm that the changes in the DNA sequence hadoccurred as designed. The sequence of version “b” of the L chain Vregion of reshaped human WS-4 antibody is shown in SEQ ID NO: 65, whilethe sequences of versions “b”, “c”, “d”, “e”, “f”, “g” and “h” of the Hchain V region of reshaped human WS-4 antibody are shown in SEQ ID Nos:41, 44, 45, 48, 51, 54 and 55, respectively.

[0187] After confirming the DNA sequences of various versions of the Vregion of reshaped human WS-4 antibody by sequence determination, theDNAs that code for the V region of reshaped human WS-4 antibody weresubcloned to mammalian cell expression vectors that already contain DNAthat codes for the human C region. Namely, DNA that codes for the Vchain L region of reshaped human WS-4 antibody was linked to a DNAsequence that codes for human L chain C region, while DNA that codes forthe H chain V region of reshaped human WS-4 antibody was linked to a DNAsequence that codes for the human Cγ1 region.

[0188] Next, all combinations of version “a” or “b” of the reshapedhuman L chain V region, and versions “a” through “h” of the H chain Vregion were tested for binding to human IL-8. As a result, as is shownin FIG. 7, both reshaped human antibodies containing L chain version “a”or “b”, and H chain version “g” (RVLa/RVHg and RVLb/RVHg) demonstratedthe ability to bind to human IL-8 to the same extent as chimeric WS-4antibody.

[0189] Any expression system, including eukaryotic cells such as animalcells or established mammalian cells, fugus cells, yeast cells andprocaryotic cells such as bacterial cells (e.g. Escherichia coli) can beused for producing the chimeric antibody or reshaped human antibodyagainst human IL-8 of the present invention. Preferably, however, thechimeric antibody or reshaped antibody of the present invention isexpressed in mammalian cells, such as COS cells or CHO cells. In thesecases, a useful, commonly used promoter can be used to express inmammalian cells. For example, it is preferable to use the humancytomegalovirus immediate early (HCMV) promoter. Examples of expressionvectors that contain HCMV promoter include HCMV-VH-HCγ1 and HCMV-VL-HCκ,as well as those derived from pSV2neo (International Patent ApplicationPublication No. W092-19759) are also included.

[0190] In addition, examples of other promoters of genetic expression inmammalian cells that can be used in the present invention that should beused include virus promoters such as retrovirus, polioma virus,adenovirus and simian virus 40 (SV40), as well as promoters originatingin mammalian cells such as human polypeptide chain elongation factor-1α(HEF-1α). For example, in the case of using SV40 promoter, expressioncan be performed by following the method of Mulligan, R. C. et al.(Nature, 277, 108-114, 1979) or in the case of using HEF-1α promoter,expression can be performed by following the method of Mizushima, S. etal. (Nucleic Acids Res., 18, 5322, 1990).

[0191] Another specific example of a useful promoter for the presentinvention is HEF-1α promoter. HEF-VH-gγ1 and HEF-VL-gκ (FIG. 1) arecontained in an expression vector containing this promoter. DNAsequences originating in polyoma virus, adenovirus, SV40 or bovinepapilloma virus (BPV) and so forth can be used as repricator points.Moreover, in order to amplify the number of genetic copies in the hostcells, aminoglucoside-3′-phosphotransferase, neo-resistant gene,thymidine kinase (TK) gene, E. coli xanthin-guaninephosphoribosyl-transferase (XGPRT) gene or dihydrofolate reductase(dhfr) can be used as selection markers.

[0192] In summary, the present invention first provides an L chain Vregion and H chain V region of mouse monoclonal antibody against humanIL-8, as well as DNA that codes for said L chain V region and DNA thatcodes for said H chain V region. These are useful in the preparation ofhuman/mouse chimeric antibody and reshaped human antibody to human IL-8.An example of monoclonal antibody is WS-4. The L chain V region has theamino acid sequence shown in, for example, SEQ ID NO: 26, while the Hchain V region has the amino acid sequence shown, for example, in SEQ IDNO: 27. These amino acid sequences are coded for by nucleotide sequencesshown, for example, in SEQ ID Nos: 26 and 27, respectively.

[0193] The chimeric antibody against human IL-8 of the present inventioncomprises:

[0194] (1) a human L chain C region and mouse L chain V region; and,

[0195] (2) a human H chain C region and mouse H chain V region.

[0196] The mouse L chain V region, mouse H chain V region and DNAs thatcode for these are as previously described. The above-mentioned human Lchain C region can be any human L chain C region, examples of whichinclude the human Cκ and Cλ regions. The above-mentioned human H chain Cregion can be any human H chain C region, examples of which include thehuman Cγ1, Cy2, Cy3 or Cy4 region (Ellison, J. et al., DNA, 1, 11-18(1981), Takahashi, N. et al., Cell, 29, 671-679 (1982), and Krawinkel,U. et al., EMBO J., 1, 403-407 (1982)).

[0197] Two types of expression vectors are prepared for producingchimeric antibody. Namely, an expression vector that contains DNA thatcodes for the mouse L chain V region and human L chain C region underthe control of an enhancer/promoter type of expression control region,and an expression vector that contains DNA that codes for the mouse Hchain V region and human H chain C region under the control of anenhancer/promoter type of expression control region. Next, host cells inthe manner of mammalian cells are simultaneously transformed with theseexpression vectors, and the transformed cells are cultured either invitro or in vivo to produce chimeric antibody.

[0198] Alternatively, DNA that codes. for mouse L chain V region andhuman L chain C region and DNA that codes for mouse H chain V region andhuman H chain C region can be introduced into a single expressionvector, host cells are transformed using said vector, and thosetransformed cells are then cultured either in vitro or in vivo toproduce chimeric antibody.

[0199] The reshaped human WS-4 antibody of the present inventioncomprises:

[0200] (A) L chains each comprising:

[0201] (1) a human L chain C region; and,

[0202] (2) an L chain V region comprising a human L chain FRs, and an Lchain CDRs of mouse monoclonal antibody WS-4 against human IL-8, as wellas

[0203] (B) H chains each comprising:

[0204] (1) a human H chain C region; and,

[0205] (2) an H chain V region comprising a human H chain FRs, and Hchain CDRs of mouse monoclonal antibody WS-4 against human IL-8.

[0206] In a preferable mode of the present invention, theabove-mentioned L chain CDR is within the amino acid sequence shown inSEQ ID NO: 26, with the extents of said amino acid sequence beingdefined in Table 5; the above-mentioned H chain CDR is within the aminoacid sequence shown in SEQ ID NO: 27, with the extents of said aminoacid sequence being defined in Table 5; the above-mentioned human Lchain FR is derived from REI; the above-mentioned human H chain FR1, FR2and FR3 are derived from VDH26, and FR4 is derived from 4B4; theabove-mentioned human L chain C region is the human Cκ region; and, theabove-mentioned human H chain C region is the human Cγ1 region. Inaddition, the above-mentioned human H chain C region may be the humanCγ4 region, or a radioisotope may be bound instead of theabove-mentioned human L chain C region and/or human H chain C region.

[0207] It is preferable to substitute a portion of the amino acidsequence of the above-mentioned human FR to prepare reshaped humanantibody that has sufficient activity with respect to a specificantigen.

[0208] In a preferable mode of the present invention, the L chain Vregion has the amino acid sequence shown as RVLa or RVLb in Table 2,while the H chain V region has the amino acid sequence shown as RVHa,RVHb, RVHc, RVHd, RVHe, RVHf, RVHg or RVHh in Tables 3 and 4. Moreover,the amino acid at position 41 in the H chain V region FR2 should beproline, the amino acid at said position 47 should be tryptophan, and/orthe amino acid at position 67 of said FR3 should be phenylalanine, andthose having the amino acid sequences shown as RVHb, RVHd, RVHe, RVHf,RVHg or RVHh are more preferable. That in which RVHg is present as the Hchain V region is the most preferable.

[0209] Two types of expression vectors are prepared for production ofreshaped antibody. Namely, an expression vector that contains DNA thatcodes for the previously defined reshaped human L chain under control byan enhancer/promoter type of expression control region, as well asanother expression vector that contains DNA that codes for thepreviously defined reshaped human H chain under control by anenhancer/promoter type of expression control region, are prepared. Next,host cells such as mammalian cells are simultaneously transformed bythese expression vectors, and the transformed cells are cultured eitherin vitro or in vivo to produce reshaped human antibody.

[0210] Alternatively, DNA that codes for reshaped human L chain and DNAthat codes for reshaped human H chain are introduced into a singleexpression vector, host cells are transformed using said vector, andthose transformed cells are then cultured either in vitro or in vivo toproduce the target reshaped human antibody.

[0211] The chimeric antibody or reshaped human antibody produced in thismanner can be isolated and purified in accordance with routine methodssuch as protein A affinity chromatography, ion exchange chromatographyor gel filtration.

[0212] The chimeric L chain or reshaped human L chain of the presentinvention can be used to prepare complete antibody by combining with anH chain. Similarly, the chimeric H chain or reshaped human H chain ofthe present invention can be used to prepare complete antibody bycombining with an L chain.

[0213] The mouse L chain V region, reshaped human L chain V region,mouse H chain V region and reshaped human H chain V region areinherently regions that bind to antigen in the form of human IL-8. Theyare considered to be useful as pharmaceuticals, diagnostic drugs and soforth either alone or in the form of fused protein with other proteins.

[0214] In addition, the L chain V region CDR and H chain V region CDR ofthe present invention are also inherently portions that bind to antigenin the form of human IL-8. These are considered to be useful aspharmaceuticals, diagnostic drugs and so forth either alone or in theform of fused protein with other proteins.

[0215] The DNA that codes for mouse L chain V region of the presentinvention is useful for preparing DNA that codes for chimeric L chain,or DNA that codes for reshaped human L chain. Similarly, the DNA thatcodes for mouse H chain V region is useful for preparing DNA that codesfor chimeric H chain or DNA that codes for reshaped human H chain. Inaddition, the DNA that codes for the L chain V region CDR of the presentinvention is useful for preparing DNA that codes for reshaped human Lchain V region, or DNA that codes for reshaped human L chain.

[0216] Similarly, the DNA that codes for the H chain V region CDR of thepresent invention is useful for preparing DNA that codes for reshapedhuman H chain V region, and DNA that codes for reshaped human H chain.Moreover, reshaped human antibody F(ab′)₂, Fab or Fv, or single chain Fvthat couples both Fv of the H chain and L chain, can be produced in asuitable host and used for the purposes described above (see, forexample, Bird, R.E. et al., TIBTECH, 9, 132-137, 1991).

[0217] Single chain Fv is composed by linking the H chain V region and Lchain V region of reshaped human antibody to human IL-8. In this singlechain Fv, the H chain V region and L chain V region are linked by alinker, and preferably a peptide linker (Huston, J.S. et al., Proc.Natl. Acad. Sci. USA, 85, 5879-5883, 1988).

[0218] The H chain V region and L chain V region of this single chain Fvmay be either of the above-mentioned H chain and L chain V regions ofreshaped human antibody. Specific examples of these include the H chainV regions composed of the amino acid sequences described in SEQ ID NOs:38, 41, 44, 45, 48, 51 and 54, and single chain Fv containing an L chainV region composed of the amino acid sequences described in SEQ ID NO: 62or 65 (see W088-01649).

[0219] These V regions are preferably linked by a peptide linker.Examples of peptide linkers that are used include any arbitrary singlechain peptide composed of, for example 12-19 residues (see W088-09344).

[0220] DNA that codes for single chain Fv is obtained by using DNA thatcodes for the H chain or H chain V region and DNA that codes for the Lchain or L chain V region of the above-mentioned reshaped human antibodyas template, amplifying the portion of DNA that codes for those aminoacid sequences that are desired using a primer pair that defines bothends by PCR, and amplifying by combining a primer pair that defines DNAthat codes for a polypeptide linker along with both its ends so as torespectively link the H and L chains.

[0221] In addition, once the DNA that code for single chain Fv areprepared, an expression vector that contains them along with a host thatis transformed by said expression vector can be obtained in accordancewith routine methods. In addition, single chain Fv can be obtained inaccordance with routine methods by using that host.

[0222] In comparison with antibody molecules, single chain Fv exhibitbetter permeability into tissue, and are expected to be used in imagingby labelling with a radioisotope, and as a therapeutic agent havingsimilar functions to reshaped human antibody.

[0223] ELISA (Enzyme-linked immunosorbent assay), EIA (Enzymeimmunoassay), RIA (radioimmunoassay) or fluorescent antibody techniquescan be used to confirm the binding activity of the chimeric antibody,reshaped human antibody and its F(ab′)₂, Fab, Fv or single chain Fvagainst IL-8 of the present invention. For example, in the case of usingenzyme immunoassay with chimeric antibody and reshaped human antibody,human IL-8 is added to a plate coated with anti-human IL-8 polyclonalantibody, a culture supernatant or purified sample of cells that producechimeric antibody or reshaped human antibody against human IL-8 isadded, and a suitable secondary antibody is added that is labeled withan enzyme such as alkaline phosphatase. After incubating and washing theplate, an enzyme substrate such as p-nitrophenylphosphate is addedfollowed by measurement of absorbance to evaluate the antigen bindingactivity.

[0224] The IL-8 binding inhibitory activity to IL-8 receptors of thechimeric antibody, reshaped human antibody, and its F(ab′)₂, Fab, Fv orsingle chain Fv against human IL-8 is evaluated by an ordinary ligandreceptor binding inhibition assay. For example, in order to assay theinhibition of binding of IL-8 to IL-8 receptors on neutrophils, afterseparating neutrophils obtained from heparinized blood by centrifugationor other means, a cell suspension is prepared having a suitable numberof cells that can be used in the above-mentioned assay.

[0225] A solution containing IL-8 suitably labeled with ¹²⁵I and soforth and non-labeled IL-8 is mixed with a solution containing theantibody of the present invention or its fragments prepared at asuitable concentration, followed by the addition of this mixture to theabove-mentioned neutrophil suspension. After a certain period of time,the neutrophils are separated, and the labeled activity on theneutrophils is assayed.

[0226] Routine known methods, such as the method described in Grob, P.M. et al., J. Biol. Chem., 265, 8311-8316, 1990, can be used forevaluation of the inhibition of neutrophil chemotaxis by the antibody orits fragments of the present invention.

[0227] In the case of using a commercially available chemotaxis chamber,after diluting the antibody or its fragments of the present inventionwith a suitable culture medium, IL-8 is added to the chamber followed bythe addition of the diluted antibody or fragments. Next, the preparedneutrophil suspension is added to the chamber and allowed to stand for acertain period of time. Since migrating neutrophils adhere to the filterinstalled in the chamber, the number of such neutrophils may be measuredby ordinary methods such as staining or fluorescent antibody methods. Inaddition, measurement may also be performed by microscopic evaluationusing a microscope or by automated measurement using a machine.

[0228] After sterilizing by filtration using a membrane filter, thechimeric antibody, reshaped human antibody and its F(ab′)₂, Fab, Fv orsingle chain Fv fragment against human IL-8 of the present invention canbe administered as a pharmaceutical therapeutic agent preferablyparenterally, by for example intravenous injection, intramuscularinjection, intraperitoneal injection or subcutaneous injection, ortranstracheally, by for example using a nebulizer. Although varyingaccording to the age and symptoms of the patient, the normal dose inhumans is 1-1000 mg/body, for which divided doses of 1-10 mg/kg/week canbe selected.

[0229] After evaluating their purified binding activity, the chimericantibody, reshaped human antibody and its F(ab′)₂, Fab, Fv or singlechain Fv fragment against human IL-8 of the present invention can beprepared into a pharmaceutical therapeutic agent by methods routinelyused for making preparations of physiologically active proteins. Forexample, a preparation for injection consists of dissolving refinedchimeric antibody, reshaped human antibody or its F(ab′)₂, Fab, Fv orsingle chain Fv fragment against human IL-8 in a a solvent such asphysiological saline or buffer, followed by the addition of ananti-adsorption agent such as Tween 80, gelatin or human serum albumin(HSA). Alternatively, this preparation may also be freeze-dried fordissolution and reconstitution prior to use. Examples of vehicles thatcan be used for freeze-drying include sugar-alcohols or sugars such asmannitol and glucose.

EXAMPLES

[0230] Although the following provides a detailed explanation of thepresent invention through its embodiments described below, the scope ofthe present invention is not limited by these examples.

Example 1

[0231] Cloning of DNA Coding for the V Region of Mouse MonoclonalAntibody against Human IL-8

[0232] DNA that codes for the variable region of mouse monoclonalantibody against human IL-8 was cloned in the manner described below.

[0233] 1. Preparation of Total RNA

[0234] Total RNA was prepared from hybridoma WS-4 by modifying thecesium chloride density gradient centrifugation method of Chirgwin, J.M. et al. described in Biochemistry, 18, 5294-5299, 1979.

[0235] Namely, 1×10⁷ hybridoma WS-4 cells were completely homogenized in25 ml of 4 M guanidine thiocyanate (Fluka). The homogenate was layeredover a 5.7 M cesium chloride solution in a centrifuge tube followed byprecipitation of the RNA by centrifuging for 14 hours at 20° C. at31,000 rpm in a Beckman SW40 rotor.

[0236] The RNA precipitate was washed with 80% ethanol and thendissolved in 200 μl of 20 mM Tris-HCl (pH 7.5) containing 10 mM EDTA and0.5% sodium N-laurylsarcosinate. After adding Protenase (Boehringer) toa concentration of 0.5 mg/ml, the resulting mixture was incubated in awater bath for 30 minutes at 37° C. The mixture was extracted withphenol and chloroform and the RNA was precipitated with ethanol. Next,the RNA precipitate was dissolved in 200 μl of 10 mM Tris-HCl (pH 7.5)containing 1 mM EDTA.

[0237] 2. Extraction of Messenger RNA (mRNA)

[0238] In order to extract mRNA coding for the H chain of mousemonoclonal antibody WS-4, poly(A)-positive mRNA was extracted from thetotal RNA obtained step 1 above using the Fast Track mRNA Isolation KitVersion 3.2 (Invitrogen) and following the procedure described in themanufacturer's instructions.

[0239] 3. Synthesis of Single Stranded cDNA

[0240] Single stranded cDNA was synthesized from approximately 40 ng ofthe mRNA obtained in step 2 above using the cDNA Cycle Kit (Invitrogen)and following the procedure described in the instructions. The resultantproduct was then used to amplify cDNA that codes for mouse H chain Vregion. Furthermore, in order to amplify cDNA that codes for mouse Lchain V region, single stranded cDNA was synthesized from approximately10 μg of the above-mentioned total RNA.

[0241] 4. Amplification of Gene Coding for Antibody Variable Region byPCR

[0242] (1) Amplication of cDNA Coding for Mouse H Chain V Region

[0243] MHV (mouse heavy variable) primers 1 to 12 shown in SEQ ID NOs:13 to 24 and MHC (mouse heavy constant) primer shown in SEQ ID NO: 25(Jones, S. T. et al., Bio/Technology, 9, 88-89, 1991) were used for thePCR primers. 100 μl of PCR solution containing 10 mM Tris-HCl (pH 8.3),50 mM KCl, 0.1 mM dNTPs (DATP, dGTP, dCTP, dTTP), 1.5 mM MgCl₂, 0.001%(w/v) gelatin, 5 units of DNA polymerase AmpliTaq (Perkin Elmer Cetus),0.25 μM of one of the MHV primers shown in SEQ ID NOs: 13 to 24, 75 μMof the MCH primer shown in SEQ ID NO: 25, and 1.5 μl of the singlestranded cDNA solution obtained in step 3 above. PCR solutions wereprepared for each of the MHV primers 1-12. After covering each solutionwith 50 μl of mineral oil, it was heated in the order of 3 minutes atthe initial temperature of 94° C., followed by a cycle of 1 minute at94° C., 1 minute at 55° C. and 1 minute at 72° C. After repeating thisheating cycle 30 times, the reaction mixture was further incubated for10 minutes at 72° C.

[0244] (2) Amplification of cDNA Coding for Mouse L Chain V Region

[0245] MKV (mouse kappa variable) primers 1 to 11 shown in SEQ ID NOs: 1to 11 and MKC (mouse kappa constant) primer shown in SEQ ID NO: 12(Jones, S.T. et al., Bio/Technology, 9, 88-89, 1991) were used for thePCR primers.

[0246] Amplification of cDNA was performed from 2.0 μl of the singlestranded cDNA obtained in step 3 above using the same method as thatdescribed for amplification of H chain V region gene in step 4 part (1)above with the exception that amplification was performed using 0.25 μMeach of the MKV primer mixtures and 3.0 μM of MCK primer.

[0247] 5. Purification and Fragmentation of PCR Product

[0248] The respective DNA fragments of the H chain V region and L chainV region amplified by PCR as described above were separated by agarosegel electrophoresis using 1.5% low melting point agarose (Sigma).Agarose pieces containing an H chain DNA fragment approximately 450 bpin length and an L chain DNA fragment approximately 400 bp in lengthwere separately cut out and melted for 5 minutes at 65° C. followed bythe addition of an equal volume of 20 mM Tris-HCl (pH 7.5) containing 2mM EDTA and 300 mM NaCl.

[0249] This mixture was extracted by phenol and chloroform, the DNAfragments were recovered by ethanol precipitation, and dissolved in 10mM Tris-HCl (pH 7.5) containing 1 mM EDTA. Next, the fragments weredigested for 3 hours at 37° C. using 5 units of restriction enzyme XmaI(New England BioLabs) in 10 mM Tris-HCl (pH 7.9) containing 10 mM MgCl₂and 1 mM dithiothreitol. Next, the DNA fragments were digested for 2hours at 37° C. with 40 units of restriction enzyme SalI (Takara Shuzo),and the resulting DNA fragments were separated by agarose gelelectrophoresis using 1.5% low melting point agarose (Sigma).

[0250] The agarose pieces containing DNA fragments were cut out andmelted for 5 minutes at 65° C. followed by the addition of an equalvolume of 20 mM Tris-HCl (pH 7.5) containing 2 mM EDTA and 300 mM NaCl.This mixture was then extracted from phenol and chloroform, the DNAfragments were recovered by ethanol precipitation and dissolved in 10 mMTris-HCl (pH 7.5) containing 1 mM EDTA.

[0251] Thus, a DNA fragment containing a gene that codes for mouseκ-type L chain V region, and a DNA fragment containing a gene that codesfor mouse H chain V region were respectively obtained. Theabove-mentioned DNA fragments both have an SalI attachment site at their5′ terminus, and an XmaI attachment site at their 3′ terminus.

[0252] 6. Linkage and Transformation

[0253] Approximately 0.3 μg of the SalI-XmaI DNA fragment containinggene that codes for mouse kappa-type L chain V region prepared in themanner described above were mixed with approximately 0.1 μg of pUC19vector (Takara Shuzo), prepared by digesting with SalI, XmaI andalkaline phosphatase of Escherichia coli (BAP; Takara Shuzo), for 4hours at 16° C. in a buffered reaction mixture containing 1 unit of T4DNA ligase (Gibco BRL) and added suplemented buffer to link.

[0254] Next, 5 μl of the above-mentioned linkage mixture were added to50 μl of competent cells of E. coli DH5α (GIBCO BRL) after which thecells were allowed to stand for 30 minutes on ice, for 1 minute at 42°C. and again for 1 minute on ice. Next, 400 μl of 2×YT medium (MolecularCloning: A Laboratory Manual, Sambrook, et al., Cold Spring HarborLaboratory Press, 1989) were added. After incubating for 1 hour at 37°C., the E. coli was spread onto 2×YT agar medium (Molecular Cloning: ALaboratory Manual, Sambrook, et al., Cold Spring Harbor LaboratoryPress, 1989) containing 50 μg/ml of ampicillin (Meiji Seika) followed byincubation overnight at 37° C. to obtain the E. coli transformant.

[0255] Subsequently, 50 μg of X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside, Takara Shuzo) were appliedas selection marker at this time.

[0256] This transformant was incubated overnight at 37° C. in 10 ml of2×YT medium containing 50 μg/ml of ampicillin, and plasmid DNA wasprepared from this culture using the QIAGEN Plasmid Mini Kit (QIAGEN)and following the procedure described in the instructions.

[0257] The plasmid containing gene that codes for mouse κ-type L chain Vregion originating in hybridoma WS-4 obtained in this manner was namedpUC-WS4-VL.

[0258] A plasmid containing gene that codes for mouse H chain V regionderived from hybridoma WS-4 was prepared from SalI-Xmal DNA fragments byfollowing the same method as described above with the exception of usingJM109 for the E. coli competent cells. The resulting plasmid was namedpUC-WS4-VH.

Example 2

[0259] Determination of DNA Nucleotide Sequence

[0260] The nucleotide sequence of the cDNA coding region in theabove-mentioned plasmids was determined using M13 Primer RV and M13Primer M4 (both Takara Shuzo) as sequence primers, an automated DNAsequencer (Applied Biosystems Inc.) and the Taq Dye Deoxy TerminatorCycle Sequencing Kit (Applied Biosystems Inc.) and following theprotocol specified by the manufacturers. The nucleotide sequence of thegene that codes for the L chain V region of mouse WS-4 antibodycontained in plasmid pUC-WS4-VL is shown in SEQ ID NO: 26. In addition,the nucleotide sequence of the gene that codes for the H chain V regionof mouse WS-4 antibody contained in plasmid pUC-WS4-VH is shown in SEQID NO: 27.

Example 3

[0261] Determination of CDR

[0262] The basic structure of the V regions of the L and H chains hasmutual similarities, each having four framework regions linked by threehyper variable regions, namely complementarity determining regions(CDR). Although the amino acid sequence of the framework region isrelatively well preserved, the variability of the amino acid sequence ofthe CDR regions is extremely high (Kabat, E. A. et al., “Sequences ofProteins of Immunological Interest”, US Dept. of Health and HumanServices, 1991).

[0263] On the basis of this fact, the CDR were determined as shown inTable 5 by investigating their homology by attempting to match the aminoacid sequence of the variable region of mouse monoclonal antibody tohuman IL-8 with the database of amino acid sequences of antibodiesprepared by Kabat, et al. TABLE 5 CDR in the L Chain V Region and HChain V Region of Mouse WS-4 Antibody Sequence Plasmid Number CDR1 CDR2CDR3 pUC-WS4-VL 26 24-34 50-56  89-97 pUC-WS4-VH 27 31-35 50-68 101-111

Example 4

[0264] Confirmation of Expression of Cloned cDNA (Preparation ofChimeric WS-4 Antibody)

[0265] Preparation of Expression Vector

[0266] In order to prepare a vector that expresses chimeric WS-4antibody, cDNA clones pUC-WS4-VL and pUC-WS4-VH, which code for the Lchain and H chain V regions of mouse WS-4, respectively, were modifiedby PCR. These were then introduced into HEF expression vector (refer tothat previously described, WO92-19759 and and FIG. 1).

[0267] The backward primer (SEQ ID NO: 28) for the L chain V region andthe backward primer (SEQ ID NO: 29) for the H chain V region wererespectively hybridized to DNA that codes for the start of the leadersequence of the V region, and designed to have a Kozak consensussequence (Kozak, M. et al., J. Mol. Biol., 196, 947-950, 1987) and aHindIII restriction site. The forward primer (SEQ ID NO: 30) for the Lchain V region and the forward primer (SEQ ID NO: 31) for the H chain Vregion were hybridized to a DNA sequence that codes for the terminal ofthe J chain, and designed to add a splice donor sequence and BamHIrestriction site. 100 μl of PCR reaction mixture containing 20 mMTris-HCl (pH 8.2), 10 mM KCl, 6 mM (NH₄)₂SO₄, 1% Triton X-100, 100 μMdNTPs, 1.5 mM MgCl₂, 100 pmoles of each primer, 100 ng of template DNA(pUC-VL or pUC-VH) and 2.5 U of AmpliTaq enzyme, were covered with 50 μlof mineral oil. After initially denaturing for 3 minutes at 94° C., aheating cycle consisting of 1 minute at 94° C., 1 minute at 55° C. and 1minute at 72° C. was repeated 30 times followed by final incubation for10 minutes at 72° C.

[0268] The PCR product was purified using 1.5% low melting point agarosegel followed by digestion with HindIII and BamHI. The L chain V regionwas cloned into HEF expression vector HEF-VL-gκ, while the H chain Vregion was cloned into HEF expression vector HEF-VH-gγ1. Afterdetermining the DNA sequences, plasmids containing the DNA fragmenthaving the correct DNA sequence were named HEF-chWS4L-gκ andHEF-chWS4H-gγ1 respectively.

[0269] Transfection into COS Cells

[0270] In order to observe the transient expression of chimeric WS-4antibody, the above-mentioned expression vectors were tested in COScells. HEF-chWS4L-gκ and HEF-chWS4H-gγ1 were simultaneously transfectedinto COS cells by electroporation using the Gene Pulser system (BioRad).Each DNA (10 μg) was added to 0.8 ml of aliquot containing 1×10⁷cells/ml in PBS, and then pulsed at 1.5 kV with a capacitance of 25 μF.

[0271] After allowing a recovery period of 10 minutes at roomtemperature, the electroporated cells were suspended in 15 ml of DMEMculture medium (GIBCO) containing 5% γ-globulin-free fetal bovine serumplaced in a tissue culture dish. After incubating for 96 hours, theculture medium was collected, cell debris were removed bycentrifugation, and the supernatant was then filtered with a disk filterhaving a pore diameter of 0.45 μm (Gelman Science).

[0272] ELISA

[0273] ELISA plates for measurement of antigen binding and antibodyconcentration were prepared as described below. The ELISA plates formeasurement of antigen binding activity were prepared in the followingmanner. After forming a solid layer in each well of a 96-well plate(Nunc) with 100 μl of goat anti-human IL-8 polyclonal antibody (R & DSystems) dissolved in a solid layer of buffer at a concentration of 2μg/ml (0.1 M sodium bicarbonate, 0.02% sodium azide), and blocking with200 μl of dilution buffer (50 mM Tris-HCl (pH 7.2), 1% bovine serumalbumin (BSA), 1 mM MgCl₂, 0.15 M NaCl, 0.05% Tween 20, and 0.02% sodiumazide), 100 μl of recombinant human IL-8 (Amersham) (5 ng/ml) was added.

[0274] A purified sample of chimeric antibody or culture supernatant ofCOS cells that expressed these was serially diluted and added to eachwell. Next, 100 μl of alkaline phosphatase-labeled goat anti-human IgGantibody (TAGO) (1 μg/ml) were added. After incubation and washing,substrate solution (1 mg/ml p-nitrophenyl-phosphate) was added followedby measurement of absorbance at 405 nm.

[0275] For measurement of antibody concentration, after forming a solidlayer in the wells of a 96-well plate with 100 μl of goat anti-human IgGantibody (TAGO) at a concentration of 1 μg/ml and blocking, a purifiedsample of chimeric antibody or culture medium of COS cells thatexpressed these was serially diluted and added to each well. Next, 100μl of alkaline phosphatase-labeled goat anti-human IgG antibody (TAGO)(1 μg/ml) was added. After incubation and washing, substrate solution (1mg/ml p-nitrophenylphosphate) was added and absorbance was measured at405 nm.

[0276] As a result, since the chimeric antibody WS-4 showed specificbinding to IL-8, it was considered that this chimeric antibody has thecorrect structure of the V region of mouse monoclonal antibody WS-4 (seeFIG. 2).

[0277] Furthermore, the Escherichia coli having above-mentioned plasmidHEF-chWS4L-gκ was deposited as Escherichia coli DH5α (HEF-chWS4L-gκ),and the Escherichia coli having the above-mentioned plasmidHEF-chWS4H-gγ1 was deposited as Escherichia coli JM109 (HEF-chWS4H-gγ1)at the Bioengineering Industrial Technology Research Institute of theAgency of Industrial Science and Technology (1-1-3 Higashi, Tsukuba,Ibaraki, Japan) on Jul. 12, 1994 under the respective names FERM BP-4739and FERM BP-4740 in accorrdance the provisions of the BudapestConvention.

[0278] Example 5

[0279] Preparation of Reshaped Human WS-4 Antibody

[0280] Preparation of the H Chain V Region of Reshaped Human WS-4Antibody

[0281] DNA that codes for the H chain V region of reshaped human WS-4antibody was designed in the manner described below. Complete DNA thatcodes for the H chain V region of reshaped human WS-4 antibody wasdesigned so that known DNA sequences that respectively code for FR1through FR3 of human antibody VDH26 and FR4 of human antibody 4B4 arelinked to the DNA sequence that codes for the CDR of the H chain Vregion of mouse WS-4 antibody.

[0282] Next, a HindIII recognition site/Kozak consensus sequence andBamHI recognition site/splice donor sequence were respectively added tothe 5′ and 3′ sides of this DNA sequence, followed by introduction intoan HEF expression vector. The DNA sequence designed in this manner wasthen divided into four approximately equal oligonucleotides after whichthe secondary structure of those oligonucleotides for which there is thepossibility of obstructing the assembly of these oligonucleotides wereanalyzed by computer.

[0283] The four oligonucleotide sequences are shown in SEQ ID NOs: 32 to35. These oligonucleotides have lengths of 113 to 143 bases, andadjacent oligonucleotides have an overlap region mutually consisting of20 bases. HF1 (SEQ ID NO: 32) and HF3 (SEQ ID NO: 34) of these fouroligonucleotides have a sense DNA sequence, while the other HF2 (SEQ IDNO: 33) and HF4 (SEQ ID NO: 35) have an antisense DNA sequence. Theseoligonucleotides were synthesized by an automated DNA synthesizer(Applied Biosystems).

[0284] In addition, the method of assembly of these fouroligonucleotides by PCR is illustrated in FIG. 3. Approximately 100 ngeach of HF1 and HF2 as well as HF3 and HF4 were combined and added to aPCR reaction mixture having a final volume of 98 μl and containing 2.5 Uof Pfu DNA polymerase. After initially denaturing for 3 minutes at 94°C., the solutions were incubated for 2 cycles each cycle consisting ofincubation for 2 minutes at 94° C., 2 minutes at 55° C. and 2 minutes at72° C.

[0285] After mutually replacing half the volume of the PCR reactionsolutions, incubation was continued for an additional two cycles. Afteradding 100 pmoles each of RVH5′ primer (SEQ ID NO: 36) and RVH3′ primer(SEQ ID NO: 37) as external primers, the PCR reaction solutions werecovered with 50 μl of mineral oil. After initially denaturing for 3minutes at 94° C., the reaction solutions were incubated for 45 cyclesof 1 minute at 94° C., 1 minute at 55° C. and 1 minute at 72° C.,followed finally by incubation for 10 minutes at 72° C.

[0286] A DNA fragment containing approximately 450 base pairs waspurified on a 1.5% low melting point agarose gel, digested with HindIIIand BamHI and cloned into HEF expression vector HEF-VH-gγ1 (FIG. 1).After determining the DNA sequence using EF-1 primer (SEQ ID NO: 66) andHIP primer (SEQ ID NO: 67), the plasmid that contained a DNA fragmentthat codes for the correct amino acid sequence of the H chain V regionwas named HEF-RVHa-gγ1. The amino acid sequence and nucleotide sequenceof the H chain V region contained in this plasmid HEF-RVHa-gγ1 are shownin SEQ ID NO: 38.

[0287] Each of the versions “b”, “c”, “d”, “e”, “f”, “g” and “h” of theH chain V region of reshaped human WS-4 antibody was prepared in themanner described below.

[0288] Version “b” (RVHb) was amplified by PCR using mutagen primersLTW1 (SEQ ID NO: 39) and LTW2 (SEQ ID NO: 40), designed so that leucineat position 47 was replaced by tryptophan, RVH5′ (SEQ ID NO: 36) andRVH3′ (SEQ ID NO: 37) for the primers that define both ends, and plasmidHEF-RVHa-gγ1 as the template DNA to obtain plasmid HEF-RVHb-gγ1. Theamino acid sequence and nucleotide sequence of the H chain V regioncontained in this plasmid HEF-RVHb-gγ1 are shown in SEQ ID NO: 41.

[0289] Version “c” was amplified by PCR using mutagen primers QTP1 (SEQID NO: 42) and QTP2 (SEQ ID NO: 43), designed so that glutamic acid atposition 41 was replaced by proline, and plasmid HEF-RVHa-gγ1 as thetemplate DNA to obtain plasmid HEF-RVHc-gγ1. The amino acid sequence andnucleotide sequence of the H chain V region contained in this plasmidHEF-RVHc-gγ1 are shown in SEQ ID NO: 44.

[0290] Version “d” was amplified by PCR using mutagen primers QTP1 andQTP2 and plasmid HEF-RVHb-gγ1 as the template DNA to obtain plasmidHEF-RVHd-gγ1. The amino acid sequence and nucleotide sequence of the Hchain V region contained in this plasmid HEF-RVHd-gγ1 are shown in SEQID NO: 45.

[0291] Version “e” was amplified by using mutagen primers ATP1 (SEQ. IDNO: 46) and ATP2 (SEQ ID NO: 47), designed so that alanine at position40 was replaced by proline, and plasmid HEF-RVHd-gγ1 as the template DNAto obtain plasmid HEF-RVHe-gγ1. The amino acid sequence and nucleotidesequence of the H chain V region contained in this plasmid HEF-RVHe-gγ1are shown in SEQ ID NO: 48.

[0292] Version “f” was amplified using mutagen primers GTA1 (SEQ ID NO:49) and GTA2 (SEQ ID NO: 50), designed so that glycine at position 44was replaced by alanine, and plasmid HEF-RVHd-gγ1 for the template DNAto obtain plasmid HEF-RVHf-gγ1. The amino acid sequence and nucleotidesequence of the H chain V region contained in this plasmid HEF-RVHf-gγ1are shown in SEQ ID NO: 51.

[0293] Version “g” was amplified using mutagen primers LTF1 (SEQ ID NO:52) and LTF2 (SEQ ID NO: 53), designed so that leucine at position 67was replaced by phenylalanine, and plasmid HEF-RVHd-gγ1 as the templateDNA to obtain plasmid HEF-RVHg-gγ1. The amino acid sequence andnucleotide sequence of the H chain V region contained in this plasmidHEF-RVHg-gγ1 are shown in SEQ ID NO: 54.

[0294] Version “h” was amplified using mutagen primers LTF1 and LTF2,and plasmid HEF-RVHb-gγ1 as the template DNA to obtain plasmidHEF-RVHh-gγ1. The amino acid sequence and nucleotide sequence of the Hchain V region contained in this plasmid HEF-RVHh-gγ1 are shown in SEQID NO: 55.

[0295] Preparation of L Chain V Region of Reshaped Human WS-4 Antibody

[0296] DNA that codes for the L chain V region of reshaped human WS-4antibody was designed in the manner described below. Complete DNA thatcodes for the L chain V region of reshaped human WS-4 antibody wasdesigned so that a DNA sequence that codes for the FR of human antibodyREI is linked to the DNA sequence that codes for the CDR of the L chainV region of mouse WS-4 antibody.

[0297] Next, a HindIII recognition site/Kozak consensus sequence andBamHI recognition site/splice donor sequence were respectively added tothe 5′ and 3′ sides of this DNA sequence so as to enable it to beintroduced into an HEF expression vector. The DNA sequence designed inthis manner was then divided into four approximately equaloligonucleotides after which the secondary structure of thoseoligonucleotides for which there is the possibility of obstructing theassembly of these oligonucleotides were analyzed by computer.

[0298] The four oligonucleotide sequences are shown in SEQ ID NOs: 56 to59. These oligonucleotides have lengths of 106 to 124 bases, andadjacent oligonucleotides have an overlap region mutually consisting of19 to 23 bases. LF1 (SEQ ID NO: 56) and LF3 (SEQ ID NO: 58) of thesefour oligonucleotides have a sense DNA sequence, while the other LF2(SEQ ID NO: 57) and LF4 (SEQ ID NO: 59) have an antisense DNA sequence.These oligonucleotides were synthesized using the same method as thatemployed for the above-mentioned HF1 through HF4.

[0299] For assembly, after initially denaturing 98 μl of a PCR mixturecontaining 100 ng of each of the four types of the nucleotides and 5 Uof Ampli Taq for 3 minutes at 94° C., the mixture was incubated for 2cycles, each cycle consisting of incubation for 2 minutes at 94° C., 2minutes at 55° C. and 2 minutes at 72° C. After adding 100 pmoles eachof RVL5′ primer (SEQ ID NO: 60) and RVL3′ primer (SEQ ID NO: 61) asexternal primers, the PCR reaction mixture was covered with 50 μl ofmineral oil. After initially denaturing for 3 minutes at 94° C., thereaction solution was incubated for 30 cycles of 1 minute at 94° C., 1minute at 550C and 1 minute at 72° C., followed finally by incubationfor 10 minutes at 72° C. (see FIG. 3).

[0300] A DNA fragment containing approximately 400 base pairs waspurified using 1.5% low melting point agarose gel, digested with HindIIIand BamHI and cloned into HEF expression vector HEF-VL-gκ (FIG. 1).After determining the DNA sequence using EF-1 primer (SEQ ID NO: 66) andKIP primer (SEQ ID NO: 68), the plasmid that contained a DNA fragmentthat codes for the correct amino acid sequence of the L chain V regionwas named HEF-RVLa-gκ. The amino acid sequence and nucleotide sequenceof the L chain V region contained in this plasmid HEF-RVLa-gκ are shownin SEQ ID NO: 62.

[0301] Version “b” (RVLb) was amplified by PCR using mutagen primersFTY1 (SEQ ID NO: 63) and FTY2 (SEQ ID NO: 64), designed so thatphenylalanine at position 71 was replaced by tyrosine, RVL5′ (SEQ ID NO:60) and RVL3′ (SEQ ID NO: 61) for the primers that define both ends, andplasmid HEF-RVLa-gκ as the template DNA to obtain plasmid HEF-RVLb-gκ.The amino acid sequence and nucleotide sequence of the L chain V regioncontained in this plasmid HEF-RVLb-gκ are shown in SEQ ID NO: 65.

[0302] In order to evaluate the antigen binding activity of each chainof the reshaped human WS-4 antibody, COS cells were first simultaneouslytransfected in the manner previously described in relation to expressionvector HEF-RVLa-gκ for version “a” of the L chain of reshaped human WS-4antibody, and expression vector HEF-chWS4H-gγ1 for the H chain ofchimeric WS-4 antibody. After collecting the culture medium aspreviously described, the amount of antibody produced and antigenbinding activity were measured for the antibodies produced using themethod described in the section on ELISA in the above Example 4. Thoseresults are shown in FIG. 4. As shown in FIG. 4, it was confirmed thatthere was no difference in antigen binding activity between chimericantibody (chL/chH), used as the positive control, and antibodyconsisting of a reshaped L chain and chimeric H chain (RVLa/chH).

[0303] At the same time, in order to evaluate the combination ofexpression vector HEF-chWS4L-gκ for the L chain of chimeric WS-4antibody and version “a” of the H chain of reshaped human WS-4 antibody,both were simultaneously CO-transfected into COS cells and the amount ofantibody produced and antigen binding activity were measured for theresulting antibody using the method described in the section on “ELISA”in the above Example 4. Antigen binding activity was not demonstratedfor this antibody (chL/RVHa) (see FIG. 4).

[0304] As previously described, since version “a” of the L chain ofreshaped human WS-4 antibody exhibited antigen binding activity equal tothat of the L chain of chimeric WS-4 antibody, evaluation of eachversion of all reshaped H chains was performed by simultaneouslytransfecting COS cells with each version of the reshaped H chain andversion “a” of the L chain of reshaped human WS-4 antibody (RVLa).

[0305] The result was that those antibodies having versions “b”, “d”,“e”, “f”, “g” and “h” of the reshaped H chain exhibited antigen bindingactivity comparable to that of chimeric WS-4 antibody (chL/chH) used asthe positive control, thus indicating that this combination forms afunctional antigen binding site in human antibody. However, with respectto the amount of antibody produced, all versions were produced in lesseramount than chimeric WS-4 antibody (chL/chH) with the exception ofversion “g” (RVHg). Furthermore, antigen binding activity was notobserved in antibody having H chain version “c” (see FIG. 5).

[0306] Based on these findings, it was concluded that antibody havingversion “a” of the L chain of reshaped human WS-4 antibody (RVLa) andversion “g” of the H chain of reshaped human WS-4 antibody reforms afunctional antigen binding site that exhibits favorable antigen bindingactivity, and that the amount of antibody produced is comparable tochimeric WS-4 antibody (chL/chH) following simultaneous transfectioninto COS cells.

[0307] Next, an evaluation of version “b” of the L chain of reshapedhuman WS-4 antibody (RVLb) was performed by simultaneously transfectingCOS cells with each version of the H chain with version “b” of the Lchain of reshaped human WS-4 antibody (RVLb). The result showed thatonly antibody having version “g” of the H chain of reshaped human WS-4antibody (RVLb/RVHg) exhibited antigen binding activity comparable tochimeric WS-4 antibody (chL/chH) used as the positive control, and itwas concluded that this combination forms a functional antigen bindingsite in human antibody. In addition, with respect to amount of antibodyproduced, all versions were produced in lesser amount than chimeric WS-4antibody (chL/chH) with the exception of version “g” (RVHg) (see FIG.6).

[0308] In the above-mentioned evaluation, the two types of reshapedhuman antibody (RVLa/RVHg and RVLb/RVHg) that exhibited binding activityto human IL-8 and extent of production comparable to that of chimericWS-4 antibody (chL/chH) were respectively purified with a Protein Acolumn, after which binding activity was evaluated accurately using themethod described in the section on ELISA in Example 4. The result showedthat chimeric WS-4 antibody (chL/chH), RVLa/RVHg antibody and RVLb/RVHgantibody all exhibited the same extents of binding activity (see FIG.7).

[0309] Based on these findings, it was concluded that antibody havingeither version “a” (RVLa) or version “b” (RVLb) of the L chain ofreshaped human WS-4 antibody and version “g” (RVHg) of the H chain ofreshaped human WS-4 antibody reforms a functional antigen binding sitethat a level of exhibits favorable antigen binding activity, and that alevel of antibody production comparable to that of chimeric WS-4antibody (chL/chH) was exhibited following simultaneous transfectioninto COS cells.

[0310] The inhibitory activity on IL-8 binding to IL-8 receptors ofreshaped human antibody consisting of version “a” (RVLa) of the H chainand version “g” (RVHg) of the H chain of reshaped human WS-4 antibody,or version “b” (RVLb) of said L chain and version “g” (RVHg) of said Hchain, was evaluated by ligand receptor binding inhibition assay.

[0311] Approximately 100 ml of heparinized blood sample from normalsubjects was layered in 35 ml aliquots onto 15 ml of Mono-Polyseparation solution (ICN Biomedicals), and the human neutrophil layerwas isolated by centrifugation according to the instructions provided.After washing these cells with RPMI-1640 medium containing 1% BSA,contaminating erythrocytes were removed with 150 mM ammonium chloridesolution. After centrifuging, the cells were washed with RPMI-1640medium containing 1% BSA and resuspended at a concentration of 2×10⁷cells/ml. The neutrophil content of this cell suspension was found to be95% or more as a result of measuring after staining smear specimensprepared using Cytospin (Shandon) with Diff-Quik stain (Green Cross).

[0312] The above-mentioned neutrophil suspension was centrifuged andresuspended at a concentration of 2×10⁷ cells/ml with binding buffer(D-PBS containing 1% BSA and 0.1% sodium azide). At this time, SK2chimeric antibody having an Fc portion identical to that of the humanantibody of the present invention (see International Patent ApplicationNo. PCT/JP94/00859) and its antigen, human IL-6, were added toconcentrations of approximately 50 μg/ml and approximately 40 ng/ml,respectively, and incubated for 30 minutes in an ice bath for thepurpose of pre-saturating the Fc receptors on the neutrophils.

[0313] IL-8 radioactively labeled with ¹²⁵I (74 TBq/mmol, Amersham) andnon-labeled IL-8 (Amersham) prepared by mixing in binding buffer atconcentrations of 4 ng/ml each. Chimeric WS-4 antibody (chL/chH),reshaped human antibody (RVLa/RVHg and RVLb/RVHg), negative controlhuman antibody (PAESEL+LOREI) or positive control mouse WS-4 antibodywas respectively diluted with binding buffer at concentrations between2000 ng/ml and approximately 8 ng/ml in stepwise, 2-fold dilutions. 50μl of IL-8 solution and 50 μl of each of the antibody solutions wereincubated for 30 minutes in an ice bath. Next, 100 μl of theabove-mentioned neutrophil suspension was added and incubation wascontinued further for 1 hour with mixing every 15 minutes. Followingincubation, the cell suspension was layered onto 200 μl of 20%saccharose solution followed by centrifugation and freezing. In order tomeasure the IL-8 bound to the cells, the cell sediment was cut away andradioactivity was measured with a gamma counter (Aroka). Those resultsare shown in FIG. 8.

[0314] Antibody having version “a” of the L chain (RVLa) and version “g”of the H chain (RVHg) of reshaped human WS-4 antibody, or version “b” ofsaid L chain and version “g” of said H chain, was clearly shown to havebinding inhibitory activity comparable to that of chimeric antibody(chL/chH) in respect of the binding of IL-8 to IL-8 receptors.

[0315] Furthermore, the Escherichia coli having the above-mentionedplasmid HEF-RVLa-gκ was deposited as Escherichia coli DH5α(HEF-RVLa-gκ), and the Escherichia coli containing plasmid HEF-RVHg-gγ1was deposited as Escherichia coli JM109 (HEF-RVHg-gγ1) at theBioengineering Industrial Technology Research Institute of the Agency ofIndustrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki,Japan) on Jul. 12, 1994 under the respective names FERM BP-4738 and FERMBP-4741 based on the provisions of the Budapest Convention.

[0316] Reference Example 1

[0317] Preparation of Hybridoma WS-4

[0318] Hybridoma that produces anti-human IL-8 monoclonal antibody wasprepared by fusing spleen cells of BALB/c mice immunized with human IL-8and mouse myeloma cells P3×63-Ag8.653 according to routine methods usingpolyethylene glycol. Screening was performed using the activity ofbinding with human IL-8 as the criterion to establish the hybridoma WS-4(Ko, Y. C. et al., J. Immunol. Methods, 149, 227-235, 1992).

[0319] INDUSTRIAL APPLICABILITY

[0320] The present invention provides reshaped human antibody againsthuman IL-8, and in this antibody, the CDR of the V region of humanantibody is substituted with the CDR of mouse monoclonal antibodyagainst human IL-8. Since the majority of this reshaped human antibodyis of human origin and CDR inherently having low antigenicity, thereshaped human antibody of the present invention has low antigenicity tohumans, and for this reason can be expected to be useful in medicaltreatment.

[0321] List of Microorganisms Deposited under the Provisions of Article13 bis of the Patent Cooperation Treaty International Deposit Authority:

[0322] Name: National Institute of Bioscience and Human-TechnologyAgency of Industrial Science and Technology

[0323] Address: 1-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan

[0324] Deposit Numbers and Deposition Dates:

[0325] (1) Escherichia coli DH5α (HEF-RVLa-gκ) Deposit no.: FERM BP-4738Deposition date: Jul. 12, 1994

[0326] (2) Escherichia coli DH5α (HEF-chWS4L-gκ) Deposit no.: FERMBP-4739 Deposition date: Jul. 12, 1994

[0327] (3) Escherichia coli JM109 (HEF-chWS4H-gγ1) Deposit no.: FERMBP-4740 Deposition date: Jul. 12, 1994

[0328] (4) Escherichia coli JM109 (HEF-RVHg-gγ1) Deposit no.: FERMBP-4741 Deposition date: Jul. 12, 1994

1 105 40 base pairs nucleic acid single linear Other 1...40 MKV1sequence 1 ACTAGTCGAC ATGAAGTTGC CTGTTAGGCT GTTGGTGCTG 40 39 base pairsnucleic acid single linear Other 1...39 MKV2 sequence 2 ACTAGTCGACATGGAGWCAG ACACACTCCT GYTATGGGT 39 40 base pairs nucleic acid singlelinear Other 1...40 MKV3 sequence 3 ACTAGTCGAC ATGAGTGTGC TCACTCAGGTCCTGGSGTTG 40 43 base pairs nucleic acid single linear Other 1...43 MKV4sequence 4 ACTAGTCGAC ATGAGGRCCC CTGCTCAGWT TYTTGGMWTC TTG 43 40 basepairs nucleic acid single linear Other 1...40 MKV5 sequence 5 ACTAGTCGACATGGATTTWC AGGTGCAGAT TWTCAGCTTC 40 37 base pairs nucleic acid singlelinear Other 1...37 MKV6 sequence 6 ACTAGTCGAC ATGAGGTKCY YTGYTSAGYTYCTGRGG 37 41 base pairs nucleic acid single linear Other 1...41 MKV7sequence 7 ACTAGTCGAC ATGGGCWTCA AGATGGAGTC ACAKWYYCWG G 41 41 basepairs nucleic acid single linear Other 1...41 MKV8 sequence 8 ACTAGTCGACATGTGGGGAY CTKTTTYCMM TTTTTCAATT G 41 35 base pairs nucleic acid singlelinear Other 1...35 MKV9 sequence 9 ACTAGTCGAC ATGGTRTCCW CASCTCAGTTCCTTG 35 37 base pairs nucleic acid single linear Other 1...37 MVK10sequence 10 ACTAGTCGAC ATGTATATAT GTTTGTTGTC TATTTCT 37 38 base pairsnucleic acid single linear Other 1...38 MVK11 sequence 11 ACTAGTCGACATGGAAGCCC CAGCTCAGCT TCTCTTCC 38 27 base pairs nucleic acid singlelinear Other 1...27 MKC sequence 12 GGATCCCGGG TGGATGGTGG GAAGATG 27 37base pairs nucleic acid single linear Other 1...37 MHV1 sequence 13ACTAGTCGAC ATGAAATGCA GCTGGGTCAT STTCTTC 37 36 base pairs nucleic acidsingle linear Other 1...36 MHV2 sequence 14 ACTAGTCGAC ATGGGATGGAGCTRTATCAT SYTCTT 36 37 base pairs nucleic acid single linear Other1...37 MHV3 sequence 15 ACTAGTCGAC ATGAAGWTGT GGTTAAACTG GGTTTTT 37 35base pairs nucleic acid single linear Other 1...35 MHV4 sequence 16ACTAGTCGAC ATGRACTTTG GGYTCAGCTT GRTTT 35 40 base pairs nucleic acidsingle linear Other 1...40 MHV5 sequence 17 ACTAGTCGAC ATGGACTCCAGGCTCAATTT AGTTTTCCTT 40 37 base pairs nucleic acid single linear Other1...37 MHV6 sequence 18 ACTAGTCGAC ATGGCTGTCY TRGSGCTRCT CTTCTGC 37 36base pairs nucleic acid single linear Other 1...36 MHV7 sequence 19ACTAGTCGAC ATGGRATGGA GCKGGRTCTT TMTCTT 36 33 base pairs nucleic acidsingle linear Other 1...33 MHV8 sequence 20 ACTAGTCGAC ATGAGAGTGCTGATTCTTTT GTG 33 40 base pairs nucleic acid single linear Other 1...40MHV9 sequence 21 ACTAGTCGAC ATGGMTTGGG TGTGGAMCTT GCTATTCCTG 40 37 basepairs nucleic acid single linear Other 1...37 MHV10 sequence 22ACTAGTCGAC ATGGGCAGAC TTACATTCTC ATTCCTG 37 38 base pairs nucleic acidsingle linear Other 1...38 MHV11 sequence 23 ACTAGTCGAC ATGGATTTTGGGCTGATTTT TTTTATTG 38 37 base pairs nucleic acid single linear Other1...37 MHV12 sequence 24 ACTAGTCGAC ATGATGGTGT TAAGTCTTCT GTACCTG 37 28base pairs nucleic acid single linear Other 1...28 MHC sequence 25GGATCCCGGG CCAGTGGATA GACAGATG 28 382 base pairs nucleic acid doublelinear cDNA mat_peptide 1...381 26 ATG AGT GTG CTC ACT CAG GTC CTG GGGTTG CTG CTG CTG TGG CTT ACA 48 Met Ser Val Leu Thr Gln Val Leu Gly LeuLeu Leu Leu Trp Leu Thr -20 -15 -10 -5 GGT GCC AGA TGT GAC ATC CAG ATGACT CAG TCT CCA GCC TCC CTA TCT 96 Gly Ala Arg Cys Asp Ile Gln Met ThrGln Ser Pro Ala Ser Leu Ser 1 5 10 GCA TCT GTG GGA GAA ACT GTC ACC ATCACA TGT CGA GCA AGT GAG ATT 144 Ala Ser Val Gly Glu Thr Val Thr Ile ThrCys Arg Ala Ser Glu Ile 15 20 25 ATT TAC AGT TAT TTA GCA TGG TAT CAG CAGAAA CAG GGA AAA TCT CCT 192 Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln LysGln Gly Lys Ser Pro 30 35 40 CAG CTC CTG GTC TAT AAT GCA AAA ACC TTA GCAGAT GGT GTG TCA TCA 240 Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala AspGly Val Ser Ser 45 50 55 60 AGG TTC AGT GGC AGT GGA TCA GGC ACA CAG TTTTCT CTG CGG ATC AGC 288 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe SerLeu Arg Ile Ser 65 70 75 AGC CTG CAG CCT GAA GAT TTT GGG AGT TAT TAC TGTCAA CAT CAT TTT 336 Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys GlnHis His Phe 80 85 90 GGT TTT CCT CGG ACG TTC GGT GGA GGC ACC AAG CTG GAACTC AAA C 382 Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu LeuLys 95 100 105 127 amino acids amino acid single linear protein internalSignal Sequence 1...20 27 Met Ser Val Leu Thr Gln Val Leu Gly Leu LeuLeu Leu Trp Leu Thr -20 -15 -10 -5 Gly Ala Arg Cys Asp Ile Gln Met ThrGln Ser Pro Ala Ser Leu Ser 1 5 10 Ala Ser Val Gly Glu Thr Val Thr IleThr Cys Arg Ala Ser Glu Ile 15 20 25 Ile Tyr Ser Tyr Leu Ala Trp Tyr GlnGln Lys Gln Gly Lys Ser Pro 30 35 40 Gln Leu Leu Val Tyr Asn Ala Lys ThrLeu Ala Asp Gly Val Ser Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly Ser GlyThr Gln Phe Ser Leu Arg Ile Ser 65 70 75 Ser Leu Gln Pro Glu Asp Phe GlySer Tyr Tyr Cys Gln His His Phe 80 85 90 Gly Phe Pro Arg Thr Phe Gly GlyGly Thr Lys Leu Glu Leu Lys 95 100 105 424 base pairs nucleic acidsingle linear mat_peptide 1...423 28 ATG AAG TTG TGG TTA AAC TGG GTT TTTCTT GTG ACA CTT TTA AAT GGT 48 Met Lys Leu Trp Leu Asn Trp Val Phe LeuVal Thr Leu Leu Asn Gly -15 -10 -5 ATC CAG TGT GAG GTG AAA CTG GTG GAGTCT GGA GGA GGC TTG ATA CAG 96 Ile Gln Cys Glu Val Lys Leu Val Glu SerGly Gly Gly Leu Ile Gln 1 5 10 CCT GGG GAT TCT CTG AGA CTC TCC TGT GTAACC TCT GGG TTC ACC TTC 144 Pro Gly Asp Ser Leu Arg Leu Ser Cys Val ThrSer Gly Phe Thr Phe 15 20 25 AGT GAT TAC TAC CTG AGC TGG GTC CGC CAG CCTCCA GGA AAG GCA CTT 192 Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Pro ProGly Lys Ala Leu 30 35 40 45 GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AATGGT TAC ACA AGA GAG 240 Glu Trp Val Gly Leu Ile Arg Asn Lys Ala Asn GlyTyr Thr Arg Glu 50 55 60 TAC AGT GCA TCT GTG AAG GGT CGG TTC ACC ATC TCCAGA GAT GAT TCC 288 Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser ArgAsp Asp Ser 65 70 75 CAA AGC ATC CTC TAT CTT CAA ATG AAC ACC CTG AGA GGTGAG GAC AGT 336 Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Gly GluAsp Ser 80 85 90 GCC ACT TAT TAC TGT GCA CGA GAG AAC TAT AGG TAC GAC GTAGAG CTT 384 Ala Thr Tyr Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Asp Val GluLeu 95 100 105 GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC TCT GCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 110 115 120 141amino acids amino acid single linear protein internal Signal Sequence1...19 29 Met Lys Leu Trp Leu Asn Trp Val Phe Leu Val Thr Leu Leu AsnGly -15 -10 -5 Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly LeuIle Gln 1 5 10 Pro Gly Asp Ser Leu Arg Leu Ser Cys Val Thr Ser Gly PheThr Phe 15 20 25 Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Pro Pro Gly LysAla Leu 30 35 40 45 Glu Trp Val Gly Leu Ile Arg Asn Lys Ala Asn Gly TyrThr Arg Glu 50 55 60 Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser ArgAsp Asp Ser 65 70 75 Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg GlyGlu Asp Ser 80 85 90 Ala Thr Tyr Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr AspVal Glu Leu 95 100 105 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val SerAla 110 115 120 34 base pairs nucleic acid single linear Other 1...34chVL backward primer 30 ACAAAGCTTC CACCATGAGT GTGCTCACTC AGGT 34 37 basepairs nucleic acid single linear Other 1...37 chVH backward primer 31GATAAGCTTC CACCATGAAG TTGTGGTTAA ACTGGGT 37 37 base pairs nucleic acidsingle linear Other 1...37 chVL forward primer 32 CTTGGATCCA CTCACGTTTGAGTTCCAGCT TGGTGCC 37 37 base pairs nucleic acid single linear Other1...37 chVH forward primer 33 GTCGGATCCA CTCACCTGCA GAGACAGTGA CCAGAGT37 137 base pairs nucleic acid single linear Other 1...137 HF1 sequence34 TAAGCTTCCA CCATGGAGTT TGGGCTGAGC TGGGTTTTCC TTGTTGCTAT TTTAAAGGG 60GTCCAGTGTG AAGTGCAGCT GTTGGAGTCT GGGGGAGGCT TGGTCCAGCC TGGGGGTT 120CTGAGACTCT CATGTGC 137 143 base pairs nucleic acid single linear Other1...143 HF2 sequence 35 GCACTGTACT CTCTTGTGTA ACCATTGGCT TTGTTTCTAATGAGACCCAC CAACTCTAG 60 CCTTTCCCTT GAGCTTGGCG GACCCAGCTC AGGTAGTAATCACTGAAGGT GAATCCAG 120 GCAGCACATG AGAGTCTCAG AGA 143 113 base pairsnucleic acid single linear Other 1...113 HF3 sequence 36 TACACAAGAGAGTACAGTGC ATCTGTGAAG GGCAGACTTA CCATCTCAAG AGAAGATTC 60 AAGAACACGCTGTATCTGCA AATGAGCAGC CTGAAAACCG AAGACTTGGC CGT 113 117 base pairsnucleic acid single linear Other 1...117 HF4 sequence 37 TCGGATCCACTCACCTGAGG AGACGGTGAC CAGGGTTCCC TGGCCCCAGT AAGCAAGCT 60 TACGTCGTAGCGATAGTTCT CTCTAGCACA GTAATACACG GCCAAGTCTT CGGTTTT 117 37 base pairsnucleic acid single linear Other 1...37 RVH5′ primer 38 GATAAGCTTCCACCATGGAG TTTGGGCTGA GCTGGGT 37 31 base pairs nucleic acid singlelinear Other 1...31 RVH3′ primer 39 GTCGGATCCA CTCACCTGAG GAGACGGTGA C31 424 base pairs nucleic acid double linear mat_peptide 1...423 40 ATGGAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48 Met GluPhe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 GTCCAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96 Val GlnCys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 CCT GGGGGT TCT CTG AGA CTC TCA TGT GCT GCC TCT GGA TTC ACC TTC 144 Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 AGT GAT TACTAC CTG AGC TGG GTC CGC CAA GCT CAA GGG AAA GGG CTA 192 Ser Asp Tyr TyrLeu Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu 30 35 40 45 GAG TTG GTGGGT CTC ATT AGA AAC AAA GCC AAT GGT TAC ACA AGA GAG 240 Glu Leu Val GlyLeu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 TAC AGT GCA TCTGTG AAG GGC AGA CTT ACC ATC TCA AGA GAA GAT TCA 288 Tyr Ser Ala Ser ValLys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser 65 70 75 AAG AAC ACG CTG TATCTG CAA ATG AGC AGC CTG AAA ACC GAA GAC TTG 336 Lys Asn Thr Leu Tyr LeuGln Met Ser Ser Leu Lys Thr Glu Asp Leu 80 85 90 GCC GTG TAT TAC TGT GCTAGA GAG AAC TAT CGC TAC GAC GTA GAG CTT 384 Ala Val Tyr Tyr Cys Ala ArgGlu Asn Tyr Arg Tyr Asp Val Glu Leu 95 100 105 GCT TAC TGG GGC CAG GGAACC CTG GTC ACC GTC TCC TCA G 424 Ala Tyr Trp Gly Gln Gly Thr Leu ValThr Val Ser Ser 110 115 120 141 amino acids amino acid single linearprotein internal Signal Sequence 1...19 41 Met Glu Phe Gly Leu Ser TrpVal Phe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 Val Gln Cys Glu Val GlnLeu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 Pro Gly Gly Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 Ser Asp Tyr Tyr Leu SerTrp Val Arg Gln Ala Gln Gly Lys Gly Leu 30 35 40 45 Glu Leu Val Gly LeuIle Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 Tyr Ser Ala Ser ValLys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser 65 70 75 Lys Asn Thr Leu TyrLeu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu 80 85 90 Ala Val Tyr Tyr CysAla Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu 95 100 105 Ala Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser 110 115 120 34 base pairs nucleicacid single linear Other 1...34 LTW1 sequence 42 GGCTAGAGTG GGTGGGTCTCATTAGAAACA AAGC 34 36 base pairs nucleic acid single linear Other 1...36LTW2 sequence 43 GAGACCCACC CACTCTAGCC CTTTCCCTTG AGCTTG 36 424 basepairs nucleic acid double linear mat_peptide 1...423 44 ATG GAG TTT GGGCTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48 Met Glu Phe Gly LeuSer Trp Val Phe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 GTC CAG TGT GAAGTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96 Val Gln Cys Glu ValGln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 CCT GGG GGT TCT CTGAGA CTC TCA TGT GCT GCC TCT GGA TTC ACC TTC 144 Pro Gly Gly Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 AGT GAT TAC TAC CTG AGCTGG GTC CGC CAA GCT CAA GGG AAA GGG CTA 192 Ser Asp Tyr Tyr Leu Ser TrpVal Arg Gln Ala Gln Gly Lys Gly Leu 30 35 40 45 GAG TGG GTG GGT CTC ATTAGA AAC AAA GCC AAT GGT TAC ACA AGA GAG 240 Glu Trp Val Gly Leu Ile ArgAsn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 TAC AGT GCA TCT GTG AAG GGCAGA CTT ACC ATC TCA AGA GAA GAT TCA 288 Tyr Ser Ala Ser Val Lys Gly ArgLeu Thr Ile Ser Arg Glu Asp Ser 65 70 75 AAG AAC ACG CTG TAT CTG CAA ATGAGC AGC CTG AAA ACC GAA GAC TTG 336 Lys Asn Thr Leu Tyr Leu Gln Met SerSer Leu Lys Thr Glu Asp Leu 80 85 90 GCC GTG TAT TAC TGT GCT AGA GAG AACTAT CGC TAC GAC GTA GAG CTT 384 Ala Val Tyr Tyr Cys Ala Arg Glu Asn TyrArg Tyr Asp Val Glu Leu 95 100 105 GCT TAC TGG GGC CAG GGA ACC CTG GTCACC GTC TCC TCA G 424 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val SerSer 110 115 120 141 amino acids amino acid single linear proteininternal Signal Sequence 1...19 45 Met Glu Phe Gly Leu Ser Trp Val PheLeu Val Ala Ile Leu Lys Gly -15 -10 -5 Val Gln Cys Glu Val Gln Leu LeuGlu Ser Gly Gly Gly Leu Val Gln 1 5 10 Pro Gly Gly Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe 15 20 25 Ser Asp Tyr Tyr Leu Ser Trp ValArg Gln Ala Gln Gly Lys Gly Leu 30 35 40 45 Glu Trp Val Gly Leu Ile ArgAsn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 Tyr Ser Ala Ser Val Lys GlyArg Leu Thr Ile Ser Arg Glu Asp Ser 65 70 75 Lys Asn Thr Leu Tyr Leu GlnMet Ser Ser Leu Lys Thr Glu Asp Leu 80 85 90 Ala Val Tyr Tyr Cys Ala ArgGlu Asn Tyr Arg Tyr Asp Val Glu Leu 95 100 105 Ala Tyr Trp Gly Gln GlyThr Leu Val Thr Val Ser Ser 110 115 120 32 base pairs nucleic acidsingle linear Other 1...32 QTP1 sequence 46 TGGGTCCGCC AAGCTCCAGGGAAAGGGCTA GA 32 32 base pairs nucleic acid single linear Other 1...32QTP2 sequence 47 TCTAGCCCTT TCCCTGGAGC TTGGCGGACC CA 32 424 base pairsnucleic acid double linear mat_peptide 1...423 48 ATG GAG TTT GGG CTGAGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48 Met Glu Phe Gly Leu SerTrp Val Phe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 GTC CAG TGT GAA GTGCAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96 Val Gln Cys Glu Val GlnLeu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 CCT GGG GGT TCT CTG AGACTC TCA TGT GCT GCC TCT GGA TTC ACC TTC 144 Pro Gly Gly Ser Leu Arg LeuSer Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 AGT GAT TAC TAC CTG AGC TGGGTC CGC CAA GCT CCA GGG AAA GGG CTA 192 Ser Asp Tyr Tyr Leu Ser Trp ValArg Gln Ala Pro Gly Lys Gly Leu 30 35 40 45 GAG TTG GTG GGT CTC ATT AGAAAC AAA GCC AAT GGT TAC ACA AGA GAG 240 Glu Leu Val Gly Leu Ile Arg AsnLys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 TAC AGT GCA TCT GTG AAG GGC AGACTT ACC ATC TCA AGA GAA GAT TCA 288 Tyr Ser Ala Ser Val Lys Gly Arg LeuThr Ile Ser Arg Glu Asp Ser 65 70 75 AAG AAC ACG CTG TAT CTG CAA ATG AGCAGC CTG AAA ACC GAA GAC TTG 336 Lys Asn Thr Leu Tyr Leu Gln Met Ser SerLeu Lys Thr Glu Asp Leu 80 85 90 GCC GTG TAT TAC TGT GCT AGA GAG AAC TATCGC TAC GAC GTA GAG CTT 384 Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr ArgTyr Asp Val Glu Leu 95 100 105 GCT TAC TGG GGC CAG GGA ACC CTG GTC ACCGTC TCC TCA G 424 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120 141 amino acids amino acid single linear protein internalSignal Sequence 1...19 49 Met Glu Phe Gly Leu Ser Trp Val Phe Leu ValAla Ile Leu Lys Gly -15 -10 -5 Val Gln Cys Glu Val Gln Leu Leu Glu SerGly Gly Gly Leu Val Gln 1 5 10 Pro Gly Gly Ser Leu Arg Leu Ser Cys AlaAla Ser Gly Phe Thr Phe 15 20 25 Ser Asp Tyr Tyr Leu Ser Trp Val Arg GlnAla Pro Gly Lys Gly Leu 30 35 40 45 Glu Leu Val Gly Leu Ile Arg Asn LysAla Asn Gly Tyr Thr Arg Glu 50 55 60 Tyr Ser Ala Ser Val Lys Gly Arg LeuThr Ile Ser Arg Glu Asp Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met SerSer Leu Lys Thr Glu Asp Leu 80 85 90 Ala Val Tyr Tyr Cys Ala Arg Glu AsnTyr Arg Tyr Asp Val Glu Leu 95 100 105 Ala Tyr Trp Gly Gln Gly Thr LeuVal Thr Val Ser Ser 110 115 120 424 base pairs nucleic acid doublelinear mat_peptide 1...423 50 ATG GAG TTT GGG CTG AGC TGG GTT TTC CTTGTT GCT ATT TTA AAG GGT 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu ValAla Ile Leu Lys Gly -15 -10 -5 GTC CAG TGT GAA GTG CAG CTG TTG GAG TCTGGG GGA GGC TTG GTC CAG 96 Val Gln Cys Glu Val Gln Leu Leu Glu Ser GlyGly Gly Leu Val Gln 1 5 10 CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GCCTCT GGA TTC ACC TTC 144 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala SerGly Phe Thr Phe 15 20 25 AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GCT CCAGGG AAA GGG CTA 192 Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Ala Pro GlyLys Gly Leu 30 35 40 45 GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AAT GGTTAC ACA AGA GAG 240 Glu Trp Val Gly Leu Ile Arg Asn Lys Ala Asn Gly TyrThr Arg Glu 50 55 60 TAC AGT GCA TCT GTG AAG GGC AGA CTT ACC ATC TCA AGAGAA GAT TCA 288 Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Ile Ser Arg GluAsp Ser 65 70 75 AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CTG AAA ACC GAAGAC TTG 336 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu AspLeu 80 85 90 GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CGC TAC GAC GTA GAGCTT 384 Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu95 100 105 GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424 AlaTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 120 141 aminoacids amino acid single linear protein internal Signal Sequence 1...1951 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly -15-10 -5 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 15 10 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 1520 25 Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 3035 40 45 Glu Trp Val Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu50 55 60 Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu80 85 90 Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu95 100 105 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115120 26 base pairs nucleic acid single linear Other 1...26 ATP1 sequence52 TGGGTCCGCC AACCTCCAGG GAAAGG 26 26 base pairs nucleic acid singlelinear Other 1...26 ATP2 sequence 53 CCTTTCCCTG GAGGTTGGCG GACCCA 26 424base pairs nucleic acid double linear mat_peptide 1...423 54 ATG GAG TTTGGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48 Met Glu Phe GlyLeu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 GTC CAG TGTGAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96 Val Gln Cys GluVal Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 CCT GGG GGT TCTCTG AGA CTC TCA TGT GCT GCC TCT GGA TTC ACC TTC 144 Pro Gly Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 AGT GAT TAC TAC CTGAGC TGG GTC CGC CAA CCT CCA GGG AAA GGG CTA 192 Ser Asp Tyr Tyr Leu SerTrp Val Arg Gln Pro Pro Gly Lys Gly Leu 30 35 40 45 GAG TGG GTG GGT CTCATT AGA AAC AAA GCC AAT GGT TAC ACA AGA GAG 240 Glu Trp Val Gly Leu IleArg Asn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 TAC AGT GCA TCT GTG AAGGGC AGA CTT ACC ATC TCA AGA GAA GAT TCA 288 Tyr Ser Ala Ser Val Lys GlyArg Leu Thr Ile Ser Arg Glu Asp Ser 65 70 75 AAG AAC ACG CTG TAT CTG CAAATG AGC AGC CTG AAA ACC GAA GAC TTG 336 Lys Asn Thr Leu Tyr Leu Gln MetSer Ser Leu Lys Thr Glu Asp Leu 80 85 90 GCC GTG TAT TAC TGT GCT AGA GAGAAC TAT CGC TAC GAC GTA GAG CTT 384 Ala Val Tyr Tyr Cys Ala Arg Glu AsnTyr Arg Tyr Asp Val Glu Leu 95 100 105 GCT TAC TGG GGC CAG GGA ACC CTGGTC ACC GTC TCC TCA G 424 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr ValSer Ser 110 115 120 141 amino acids amino acid single linear proteininternal Signal Sequence 1...19 55 Met Glu Phe Gly Leu Ser Trp Val PheLeu Val Ala Ile Leu Lys Gly -15 -10 -5 Val Gln Cys Glu Val Gln Leu LeuGlu Ser Gly Gly Gly Leu Val Gln 1 5 10 Pro Gly Gly Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe 15 20 25 Ser Asp Tyr Tyr Leu Ser Trp ValArg Gln Pro Pro Gly Lys Gly Leu 30 35 40 45 Glu Trp Val Gly Leu Ile ArgAsn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 Tyr Ser Ala Ser Val Lys GlyArg Leu Thr Ile Ser Arg Glu Asp Ser 65 70 75 Lys Asn Thr Leu Tyr Leu GlnMet Ser Ser Leu Lys Thr Glu Asp Leu 80 85 90 Ala Val Tyr Tyr Cys Ala ArgGlu Asn Tyr Arg Tyr Asp Val Glu Leu 95 100 105 Ala Tyr Trp Gly Gln GlyThr Leu Val Thr Val Ser Ser 110 115 120 29 base pairs nucleic acidsingle linear Other 1...29 GTA1 sequence 56 CAAGCTCCAG GGAAAGCGCTAGAGTGGGT 29 29 base pairs nucleic acid single linear Other 1...29 GTA2sequence 57 ACCCACTCTA GCGCTTTCCC TGGAGCTTG 29 424 base pairs nucleicacid double linear mat_peptide 1...423 58 ATG GAG TTT GGG CTG AGC TGGGTT TTC CTT GTT GCT ATT TTA AAG GGT 48 Met Glu Phe Gly Leu Ser Trp ValPhe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 GTC CAG TGT GAA GTG CAG CTGTTG GAG TCT GGG GGA GGC TTG GTC CAG 96 Val Gln Cys Glu Val Gln Leu LeuGlu Ser Gly Gly Gly Leu Val Gln 1 5 10 CCT GGG GGT TCT CTG AGA CTC TCATGT GCT GCC TCT GGA TTC ACC TTC 144 Pro Gly Gly Ser Leu Arg Leu Ser CysAla Ala Ser Gly Phe Thr Phe 15 20 25 AGT GAT TAC TAC CTG AGC TGG GTC CGCCAA GCT CCA GGG AAA GCG CTA 192 Ser Asp Tyr Tyr Leu Ser Trp Val Arg GlnAla Pro Gly Lys Ala Leu 30 35 40 45 GAG TGG GTG GGT CTC ATT AGA AAC AAAGCC AAT GGT TAC ACA AGA GAG 240 Glu Trp Val Gly Leu Ile Arg Asn Lys AlaAsn Gly Tyr Thr Arg Glu 50 55 60 TAC AGT GCA TCT GTG AAG GGC AGA CTT ACCATC TCA AGA GAA GAT TCA 288 Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr IleSer Arg Glu Asp Ser 65 70 75 AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CTGAAA ACC GAA GAC TTG 336 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu LysThr Glu Asp Leu 80 85 90 GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CGC TACGAC GTA GAG CTT 384 Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr AspVal Glu Leu 95 100 105 GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCCTCA G 424 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115120 141 amino acids amino acid single linear protein internal SignalSequence 1...19 59 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala IleLeu Lys Gly -15 -10 -5 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly GlyGly Leu Val Gln 1 5 10 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala SerGly Phe Thr Phe 15 20 25 Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Ala ProGly Lys Ala Leu 30 35 40 45 Glu Trp Val Gly Leu Ile Arg Asn Lys Ala AsnGly Tyr Thr Arg Glu 50 55 60 Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr IleSer Arg Glu Asp Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser LeuLys Thr Glu Asp Leu 80 85 90 Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr ArgTyr Asp Val Glu Leu 95 100 105 Ala Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser 110 115 120 23 base pairs nucleic acid single linear Other1...23 LTF1 sequence 60 GTGAAGGGCA GATTTACCAT CTC 23 23 base pairsnucleic acid single linear Other 1...23 LTF2 sequence 61 GAGATGGTAAATCTGCCCTT CAC 23 424 base pairs nucleic acid double linear mat_peptide1...423 62 ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAGGGT 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-15 -10 -5 GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTCCAG 96 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 15 10 CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GCC TCT GGA TTC ACC TTC 144Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GCT CCA GGG AAA GGG CTA 192 SerAsp Tyr Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 30 35 40 45GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AAT GGT TAC ACA AGA GAG 240 GluTrp Val Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 TACAGT GCA TCT GTG AAG GGC AGA TTT ACC ATC TCA AGA GAA GAT TCA 288 Tyr SerAla Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asp Ser 65 70 75 AAG AACACG CTG TAT CTG CAA ATG AGC AGC CTG AAA ACC GAA GAC TTG 336 Lys Asn ThrLeu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu 80 85 90 GCC GTG TATTAC TGT GCT AGA GAG AAC TAT CGC TAC GAC GTA GAG CTT 384 Ala Val Tyr TyrCys Ala Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu 95 100 105 GCT TAC TGGGGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424 Ala Tyr Trp Gly Gln GlyThr Leu Val Thr Val Ser Ser 110 115 120 141 amino acids amino acidsingle linear protein internal Signal Sequence 1...19 63 Met Glu Phe GlyLeu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 Val Gln CysGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 Ser Asp TyrTyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 30 35 40 45 Glu TrpVal Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 Tyr SerAla Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asp Ser 65 70 75 Lys AsnThr Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu 80 85 90 Ala ValTyr Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu 95 100 105 AlaTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 120 424 basepairs nucleic acid double linear mat_peptide 1...423 64 ATG GAG TTT GGGCTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48 Met Glu Phe Gly LeuSer Trp Val Phe Leu Val Ala Ile Leu Lys Gly -15 -10 -5 GTC CAG TGT GAAGTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96 Val Gln Cys Glu ValGln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 CCT GGG GGT TCT CTGAGA CTC TCA TGT GCT GCC TCT GGA TTC ACC TTC 144 Pro Gly Gly Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 AGT GAT TAC TAC CTG AGCTGG GTC CGC CAA GCT CAA GGG AAA GGG CTA 192 Ser Asp Tyr Tyr Leu Ser TrpVal Arg Gln Ala Gln Gly Lys Gly Leu 30 35 40 45 GAG TGG GTG GGT CTC ATTAGA AAC AAA GCC AAT GGT TAC ACA AGA GAG 240 Glu Trp Val Gly Leu Ile ArgAsn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 TAC AGT GCA TCT GTG AAG GGCAGA TTT ACC ATC TCA AGA GAA GAT TCA 288 Tyr Ser Ala Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Glu Asp Ser 65 70 75 AAG AAC ACG CTG TAT CTG CAA ATGAGC AGC CTG AAA ACC GAA GAC TTG 336 Lys Asn Thr Leu Tyr Leu Gln Met SerSer Leu Lys Thr Glu Asp Leu 80 85 90 GCC GTG TAT TAC TGT GCT AGA GAG AACTAT CGC TAC GAC GTA GAG CTT 384 Ala Val Tyr Tyr Cys Ala Arg Glu Asn TyrArg Tyr Asp Val Glu Leu 95 100 105 GCT TAC TGG GGC CAG GGA ACC CTG GTCACC GTC TCC TCA G 424 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val SerSer 110 115 120 141 amino acids amino acid single linear proteininternal Signal Sequence 1...19 65 Met Glu Phe Gly Leu Ser Trp Val PheLeu Val Ala Ile Leu Lys Gly -15 -10 -5 Val Gln Cys Glu Val Gln Leu LeuGlu Ser Gly Gly Gly Leu Val Gln 1 5 10 Pro Gly Gly Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe 15 20 25 Ser Asp Tyr Tyr Leu Ser Trp ValArg Gln Ala Gln Gly Lys Gly Leu 30 35 40 45 Glu Trp Val Gly Leu Ile ArgAsn Lys Ala Asn Gly Tyr Thr Arg Glu 50 55 60 Tyr Ser Ala Ser Val Lys GlyArg Phe Thr Ile Ser Arg Glu Asp Ser 65 70 75 Lys Asn Thr Leu Tyr Leu GlnMet Ser Ser Leu Lys Thr Glu Asp Leu 80 85 90 Ala Val Tyr Tyr Cys Ala ArgGlu Asn Tyr Arg Tyr Asp Val Glu Leu 95 100 105 Ala Tyr Trp Gly Gln GlyThr Leu Val Thr Val Ser Ser 110 115 120 124 base pairs nucleic acidsingle linear Other 1...124 LF1 sequence 66 TTGAAGCTTC CACCATGGGATGGAGCTGTA TCATCCTCTT CTTGGTAGCA ACAGCTACA 60 GTGTCCACTC CGACATCCAGATGACCCAGA GCCCAAGCAG CCTGAGCGCC AGCGTAGG 120 ACAG 124 122 base pairsnucleic acid single linear Other 1...122 LF2 sequence 67 GCATTGTAGATCAGCAGCTT TGGAGCCTTT CCTGGCTTCT GCTGGTACCA TGCTAAATA 60 CTGTAAATAATCTCGCTTGC TCGACAGGTG ATGGTCACTC TGTCACCTAC GCTGGCGC 120 AG 122 121 basepairs nucleic acid single linear Other 1...121 LF3 sequence 68AGCTGCTGAT CTACAATGCA AAAACCTTAG CAGATGGAGT GCCAAGCAGA TTCAGCGGT 60GCGGTAGCGG TACCGACTTC ACCTTCACCA TCAGCAGCCT CCAGCCAGAG GACATCGC 120 C121 106 base pairs nucleic acid single linear Other 1...106 LF4 sequence69 GTAGGATCCA CTCACGTTTG ATTTCGACCT TGGTCCCTTG GCCGAACGTC CGAGGAAAA 60CAAAATGATG TTGGCAGTAG TAGGTAGCGA TGTCCTCTGG CTGGAG 106 20 base pairsnucleic acid single linear Other 1...20 RVL5′ sequence 70 TTGAAGCTTCCACCATGGGA 20 20 base pairs nucleic acid single linear Other 1...20RVL3′ sequence 71 GTAGGATCCA CTCACGTTTG 20 379 base pairs nucleic aciddouble linear mat_peptide 1...378 72 ATG GGA TGG AGC TGT ATC ATC CTC TTCTTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Phe LeuVal Ala Thr Ala Thr Gly -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAGAGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala 1 5 10 AGC GTA GGT GAC AGA GTG ACC ATC ACC TGTCGA GCA AGC GAG ATT ATT 144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys ArgAla Ser Glu Ile Ile 15 20 25 TAC AGT TAT TTA GCA TGG TAC CAG CAG AAG CCAGGA AAG GCT CCA AAG 192 Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 30 35 40 45 CTG CTG ATC TAC AAT GCA AAA ACC TTA GCA GATGGA GTG CCA AGC AGA 240 Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala Asp GlyVal Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTCACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe ThrIle Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCT ACC TAC TAC TGC CAA CATCAT TTT GGT 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His HisPhe Gly 80 85 90 TTT CCT CGG ACG TTC GGC CAA GGG ACC AAG GTC GAA ATC AAAC 379 Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105126 amino acids amino acid single linear protein internal SignalSequence 1...19 73 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala ThrAla Thr Gly -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg AlaSer Glu Ile Ile 15 20 25 Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala AspGly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrPhe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr CysGln His His Phe Gly 80 85 90 Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys 95 100 105 38 base pairs nucleic acid single linear Other1...38 FTY1 sequence 74 AGCGGTAGCG GTACCGACTA CACCTTCACC ATCAGCAG 38 38base pairs nucleic acid single linear Other 1...38 FTY2 sequence 75CTGCTGATGG TGAAGGTGTA GTCGGTACCG CTACCGCT 38 379 base pairs nucleic aciddouble linear mat_peptide 1...378 76 ATG GGA TGG AGC TGT ATC ATC CTC TTCTTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Phe LeuVal Ala Thr Ala Thr Gly -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAGAGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala 1 5 10 AGC GTA GGT GAC AGA GTG ACC ATC ACC TGTCGA GCA AGC GAG ATT ATT 144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys ArgAla Ser Glu Ile Ile 15 20 25 TAC AGT TAT TTA GCA TGG TAC CAG CAG AAG CCAGGA AAG GCT CCA AAG 192 Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 30 35 40 45 CTG CTG ATC TAC AAT GCA AAA ACC TTA GCA GATGGA GTG CCA AGC AGA 240 Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala Asp GlyVal Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TAC ACC TTCACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe ThrIle Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCT ACC TAC TAC TGC CAA CATCAT TTT GGT 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His HisPhe Gly 80 85 90 TTT CCT CGG ACG TTC GGC CAA GGG ACC AAG GTC GAA ATC AAAC 379 Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105126 amino acids amino acid single linear protein internal SignalSequence 1...19 77 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala ThrAla Thr Gly -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg AlaSer Glu Ile Ile 15 20 25 Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala AspGly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr ThrPhe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr CysGln His His Phe Gly 80 85 90 Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys 95 100 105 18 base pairs nucleic acid single linear Other1...18 EF1 sequence 78 CAGACAGTGG TTCAAAGT 18 17 base pairs nucleic acidsingle linear Other 1...17 HIP sequence 79 GCCCCAAAGC CAAGGTC 17 20 basepairs nucleic acid single linear Other 1...20 KIP sequence 80 AACTCAATGCTTTAGGCAAA 20 11 amino acids amino acid single linear 81 Arg Ala Ser GluIle Ile Tyr Ser Tyr Leu Ala 1 5 10 7 amino acids amino acid singlelinear 82 Asn Ala Lys Thr Leu Ala Asp 1 5 9 amino acids amino acidsingle linear 83 Gln His His Phe Gly Phe Pro Arg Thr 1 5 5 amino acidsamino acid single linear 84 Asp Tyr Tyr Leu Ser 1 5 19 amino acids aminoacid single linear 85 Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg GluTyr Ser Ala Ser 1 5 10 15 Val Lys Gly 11 amino acids amino acid singlelinear 86 Glu Asn Tyr Arg Tyr Asp Val Glu Leu Ala Tyr 1 5 10 23 aminoacids amino acid single linear 87 Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 15amino acids amino acid single linear 88 Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys Leu Leu Ile Tyr 1 5 10 15 32 amino acids amino acid singlelinear 89 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr 1 5 10 15 Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys 20 25 30 10 amino acids amino acid single linear 90 Phe Gly GlnGly Thr Lys Val Glu Ile Lys 1 5 10 32 amino acids amino acid singlelinear 91 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp TyrThr 1 5 10 15 Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys 20 25 30 30 amino acids amino acid single linear 92 Glu Val GlnLeu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 14 amino acidsamino acid single linear 93 Trp Val Arg Gln Ala Gln Gly Lys Gly Leu GluLeu Val Gly 1 5 10 32 amino acids amino acid single linear 94 Arg LeuThr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 MetSer Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg 20 25 30 11amino acids amino acid single linear 95 Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser 1 5 10 14 amino acids amino acid single linear 96 Trp ValArg Gln Ala Gln Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 14 amino acidsamino acid single linear 97 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluLeu Val Gly 1 5 10 14 amino acids amino acid single linear 98 Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 14 amino acidsamino acid single linear 99 Trp Val Arg Gln Pro Pro Gly Lys Gly Leu GluTrp Val Gly 1 5 10 14 amino acids amino acid single linear 100 Trp ValArg Gln Ala Pro Gly Lys Ala Leu Glu Trp Val Gly 1 5 10 14 amino acidsamino acid single linear 101 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val Gly 1 5 10 32 amino acids amino acid single linear 102 Arg PheThr Ile Ser Arg Glu Asp Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 MetSer Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Ala Arg 20 25 30 14amino acids amino acid single linear 103 Trp Val Arg Gln Ala Gln Gly LysGly Leu Glu Trp Val Gly 1 5 10 80 amino acids amino acid single linear104 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 510 15 Asp Arg Val Thr Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly Lys Ala 2025 30 Pro Lys Leu Leu Ile Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 3540 45 Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp 5055 60 Ile Ala Thr Tyr Tyr Cys Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 6570 75 80 87 amino acids amino acid single linear 105 Glu Val Gln Leu LeuGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Val 20 25 30 Arg Gln Ala GlnGly Lys Gly Leu Glu Leu Val Gly Arg Leu Thr Ile 35 40 45 Ser Arg Glu AspSer Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu 50 55 60 Lys Thr Glu AspLeu Ala Val Tyr Tyr Cys Ala Arg Trp Gly Gln Gly 65 70 75 80 Thr Leu ValThr Val Ser Ser 85

1. A light chain (L chain) variable region (V region) of mousemonoclonal antibody against human Interleukin-8 (IL-8).
 2. An L chain Vregion as set forth in claim 1 having the amino acid sequence or aportion thereof shown in SEQ ID NO:
 26. 3. A heavy chain (H chain) Vregion of mouse monoclonal antibody against human IL-8.
 4. An H chain Vregion as set forth in claim 3 having the amino acid sequence or aportion thereof shown in SEQ ID NO:
 27. 5. Chimeric L chain comprising ahuman L chain constant region (C region), and an L chain V region ofmouse monoclonal antibody against human IL-8.
 6. Chimeric L chain as setforth in claim 5, wherein said mouse L chain V region has the amino acidsequence or a portion thereof shown in SEQ ID NO:
 26. 7. Chimeric Hchain comprising a human H chain C region, and an H chain V region ofmouse monoclonal antibody against human IL-8.
 8. Chimeric H chain as setforth in claim 7, wherein said mouse H chain V-region has the amino acidsequence or a portion thereof shown in SEQ ID NO:
 27. 9. Chimericantibody comprising: (1) L chains each comprising a human L chainconstant region (C region), and an L chain V region of mouse monoclonalantibody against human IL-8; and, (2) H chains each comprising a human Hchain C region, and an H chain V region of mouse monoclonal antibodyagainst human IL-8.
 10. Chimeric antibody as set forth in claim 9wherein said mouse L chain V region has the amino acid sequence or aportion thereof shown in SEQ ID NO: 26, and said mouse H chain V regionhas the amino acid sequence or a portion thereof shown in SEQ ID NO: 27.11. A complementarity determining region (CDR) of an L chain V region ofmouse monoclonal antibody against human IL-8.
 12. A CDR as set forth inclaim 11 having the amino acid sequence or a portion thereof shown belowand in SEQ ID NO:
 26. CDR1: Arg Ala Ser/Glu Ile Ile/Tyr Ser Tyr/Leu Ala/CDR2: Asn Ala Lys/Thr Leu Ala/Asp CDR3: Gln His His/Phe Gly Phe/Pro ArgThr/
 13. A CDR of an H chain V region of mouse monoclonal antibodyagainst human IL-8.
 14. A CDR as set forth in claim 13 having the aminoacid sequence or a portion thereof shown below and in SEQ ID NO: 27.CDR1: Asp Tyr Tyr/Leu Ser CDR2: Leu Ile Arg/Asn Lys Ala/Asn Gly Tyr/ThrArg Glu/ Tyr Ser Ala/Ser Val Lys/Gly CDR3: Glu Asn Tyr/Arg Tyr Asp/ValGlu Leu/Ala Tyr/
 15. A reshaped human L chain V region of antibodyagainst human IL-8 comprising: (1) a framework region (FR) of a human Lchain V region; and, (2) a CDR of an L chain V region of mousemonoclonal antibody against human IL-8.
 16. A reshaped human L chain Vregion as set forth in claim 15, wherein said CDR has the amino acidsequence or a portion thereof shown in claim
 12. 17. A reshaped human Lchain V region as set forth in claims 15 and 16, wherein said FR isderived from human antibody REI.
 18. A reshaped human L chain V regionas set forth in claim 15, wherein said L chain V region has the aminoacid sequence or a portion thereof indicated as RVLa or RVLb in Table 2.19. A reshaped human H chain V region of antibody against human IL-8comprising: (1) an FR of a human H chain V region; and, (2) a CDR of anH chain V region of mouse monoclonal antibody against human IL-8.
 20. Areshaped human H chain V region as set forth in claim 19, wherein saidCDR has the amino acid sequence or a portion thereof shown in claim 14.21. A reshaped human H chain V region as set forth in claims 19 and 20,wherein said FR1, FR2 and FR3 are derived from human antibody VDH26 andsaid FR4 is derived from human antibody 4B4.
 22. A reshaped human Hchain V region as set forth in claim 19, wherein said H chain V regionhas the amino acid sequence or a portion thereof indicated as RVHa,RVHb, RVHc, RVHd, RVHe, RVHf, RVHg or RVHh in Tables 3 and
 4. 23. An Lchain of reshaped human antibody against human IL-8 comprising: (1) ahuman L chain C region; and, (2) an L chain V region comprising a humanL chain FR, and an L chain CDR of mouse monoclonal antibody againsthuman IL-8.
 24. An L chain of reshaped human antibody as set forth inclaim 23, wherein said human L chain C region is a human Cκ region,human L chain FR is derived from human antibody REI, and said L chainCDR has the amino acid sequence or a portion thereof shown in claim 12.25. An L chain of reshaped human antibody as set forth in claim 23,wherein said L chain V region has the amino acid sequence or a portionthereof indicated as RVLa or RVLb in Table
 2. 26. An H chain of reshapedhuman antibody against human IL-8 comprising: (1) a human H chain Cregion; and, (2) an H chain V region comprising a human H chain FR, andan H chain CDR of mouse monoclonal antibody against human IL-8.
 27. An Hchain of reshaped human antibody as set forth in claim 26, wherein saidhuman H chain C region is a human Cγ1 region, said human H chain FR1,FR2 and FR3 are derived from human antibody VDH26,.human H chain FR4 isderived from human antibody 4B4, and said H chain CDR has the amino acidsequence or a portion thereof shown in claim
 14. 28. An H chain ofreshaped human antibody as set forth in claim 26, wherein said H chain Vregion has the amino acid sequence or a portion thereof indicated asRVHa, RVHb, RVHc, RVHd, RVHe, RVHf, RVHg and RVHh Tables 3 and
 4. 29. Areshaped human antibody against human IL-8 comprising: (A) L chains eachcomprising: (1) a human L chain C region; and, (2) an L chain V regioncomprising a human L chain FR, and L chain CDR of mouse monoclonalantibody against human IL-8; and, (B) an H chain comprising: (1) a humanH chain C region; and, (2) an H chain V region comprising a human Hchain FR, and H chain CDR of mouse monoclonal antibody against humanIL-8.
 30. A reshaped human antibody as set forth in claim 29, whereinsaid L chain CDR has the amino acid sequence or a portion thereof shownin claim 12, and said H chain CDR has the amino acid sequence or aportion thereof shown in claim
 14. 31. A reshaped human antibody as setforth in claim 29, wherein said L chain CDR has the amino acid sequenceor a portion thereof shown in claim 12, and said H chain CDR has theamino acid sequence or a portion thereof shown in claim 14; said human Lchain FR is derived from human antibody REI; said human H chain FR1, FR2and FR3 are derived from human antibody VDH26, human H chain FR4 isderived from human antibody 4B4, and said human L chain C region is ahuman Cκ region; and, said human H chain C region is a human Cγ1 region.32. A reshaped human antibody as set forth in claim 29, wherein said Lchain CDR has the amino acid sequence or a portion thereof shown inclaim 12, and said H chain CDR has the amino acid sequence or a portionthereof shown in claim 14; said human L chain FR is derived from humanantibody REI; said human H chain FR1, FR2 and FR3 are derived from humanantibody VDH26, human H chain FR4 is derived from human antibody 4B4,and said human L chain C region is a human Cκ region; and, said human Hchain C region is Cγ4.
 33. A reshaped human antibody as set forth inclaim 29 wherein said L chain V region has the amino acid sequence or aportion thereof indicated as RVLa or RVLb in Table
 2. 34. A reshapedhuman antibody as set forth in claim 29, wherein said H chain V regionhas the amino acid sequence or a portion thereof indicated as RVHa,RVHb, RVHc, RVHd, RVHe, RVHf, RVHg or RVHh in Tables 3 and
 4. 35. DNAthat codes for a chimeric L chain of antibody against human IL-8containing: (1) a human L chain C region; and, (2) an L chain V regionof mouse monoclonal antibody against human IL-8.
 36. DNA as set forth inclaim 35, wherein said L chain V region codes for the amino acidsequence or a portion thereof shown in SEQ ID NO:
 26. 37. DNA as setforth in claim 35, wherein the DNA that codes for said L chain V regionhas the nucleotide sequence or a portion thereof shown in SEQ ID NO: 26.38. DNA that codes for a chimeric H chain of antibody against human IL-8containing: (1) a human H chain C region; and, (2) an H chain V regionof mouse monoclonal antibody against human IL-8.
 39. DNA as set forth inclaim 38, wherein said H chain V region has the amino acid sequence or aportion thereof shown in SEQ ID NO:
 27. 40. DNA as set forth in claim38, wherein said H chain V region has the nucleotide sequence or aportion thereof shown in SEQ ID NO:
 27. 41. DNA that codes for an Lchain V region of mouse monoclonal antibody against human IL-8.
 42. DNAas set forth in claim 41, wherein said L chain V region codes for theamino acid sequence or a portion thereof shown in SEQ ID NO:
 26. 43. DNAas set forth in claim 41, wherein the DNA that codes for said L chain Vregion has the nucleotide sequence of a portion thereof shown in SEQ IDNO:
 26. 44. DNA that codes for an H chain V region of mouse monoclonalantibody against human IL-8.
 45. DNA as set forth in claim 44, whereinsaid H chain V region codes for the amino acid sequence or a portionthereof shown in SEQ ID NO:
 27. 46. DNA as set forth in claim 44,wherein the DNA that codes for said H chain V region has the nucleotidesequence or a portion thereof shown in SEQ ID NO:
 27. 47. DNA that codesfor CDR of an L chain V region of mouse monoclonal antibody againsthuman IL-8.
 48. DNA that codes for CDR as set forth in claim 47, whereinsaid CDR codes for the amino acid sequence or a portion thereof shown inclaim
 12. 49. DNA that codes for CDR as set forth in claim 47, whereinsaid CDR has the nucleotide sequence or a portion thereof shown in SEQID NO:
 26. 50. DNA that codes for CDR of an H chain V region of mousemonoclonal antibody against human IL-8.
 51. DNA that codes for CDR asset forth in claim 50, wherein said CDR has the amino acid sequence or aportion thereof shown in claim
 14. 52. DNA that codes for CDR as setforth in claim 50, wherein said CDR has the nucleotide sequence or aportion thereof shown in SEQ ID NO:
 27. 53. DNA that codes for areshaped human L chain V region of antibody against human IL-8comprising: (1) FR of a human L chain V region; and, (2) CDR of an Lchain V region of mouse monoclonal antibody against human IL-8.
 54. DNAthat codes for a reshaped human L chain V region as set forth in claim53, wherein said CDR has the amino acid sequence or a portion thereofshown in claim
 12. 55. DNA that codes for a reshaped human L chain Vregion as set forth in claims 53 and 54, wherein said FR is derived fromhuman antibody REI.
 56. DNA as set forth in claim 53, wherein said Lchain V region codes for the amino acid sequence or a portion thereofindicated as RVLa or RVLb in Table
 2. 57. DNA as set forth in claim 53having the nucleotide sequence of a portion thereof shown in SEQ ID NO:62 or SEQ ID NO:
 65. 58. DNA that codes for a reshaped human H chain Vregion of antibody against human IL-8 comprising: (1) FR of a human Hchain V region; and, (2) CDR of an H chain V region of mouse monoclonalantibody against human IL-8.
 59. DNA that codes for a reshaped human Hchain V region as set forth in claim 58, wherein said CDR has the aminoacid sequence or a portion thereof shown in claim
 14. 60. DNA that codesfor a reshaped human H chain V region as set forth in claims 58 and 59,wherein said FR1, FR2 and FR3 is derived from human antibody VDH26, andFR4 are derived from human antibody 4B4.
 61. DNA that codes for areshaped human H chain V region as set forth in claim 58, wherein said Hchain V region codes for the amino acid sequence or a portion thereofindicated as RVHa, RVHb, RVHc, RVHd, RVHe, RVHf, RVHg or RVHh in Tables3 and
 4. 62. DNA as set forth in claim 48 having the nucleotide sequenceor a portion thereof shown in SEQ ID NO: 38, 41, 44, 45, 48, 51, 54 or55.
 63. DNA that codes for a reshaped human L chain of antibody againsthuman IL-8 comprising: (1) a human L chain C region; and, (2) an L chainV region comprising human FR, and CDR of mouse monoclonal antibodyagainst human IL-8.
 64. DNA as set forth in claim 63, wherein said Lchain V region codes for the amino acid sequence or a portion thereofindicated as RVLa or RVLb in Table
 2. 65. DNA as set forth in claim 63,wherein said L chain V region has the nucleotide sequence or a portionthereof shown in SEQ ID NO: 62 or SEQ ID NO:
 65. 66. DNA as set forth inclaims 63, 64 and 65, wherein said human L chain C region is a human Lchain Cκ region.
 67. DNA that codes for a reshaped human H chain ofantibody against human IL-8 comprising: (1) a human H chain C region;and, (2) an H chain V region comprising human FR, and CDR of mousemonoclonal antibody against human IL-8.
 68. DNA that codes for reshapedhuman H chain as set forth in claim 67 wherein said H chain V regioncodes for the amino acid sequence or a portion thereof indicated asRVHa, RVHb, RVHc, RVHd, RVHe, RVHf, RVHg or RVHh in Tables 3 and
 4. 69.DNA as set forth in claim 67, wherein said H chain V region has thenucleotide sequence or a portion thereof shown in SEQ ID NO: 38, 41, 44,45, 48, 51, 54 or
 55. 70. DNA as set forth in claims 67, 68 or 69,wherein said human H chain C region is a human H chain Cγ1 region. 71.DNA as set forth in claims 67, 68 or 69, wherein said human H chain Cregion is a human H chain Cγ4 region.
 72. A vector containing DNA as setforth in any one of claims 35, 36, 37, 38, 39, 40, 63, 64, 65, 66, 67,68, 69, 70 and
 71. 73. A host cell transformed by a vector as set forthin claim
 72. 74. A process for producing chimeric antibody against humanIL-8 comprising the steps of culturing host cells transformedsimultaneously with an expression vector containing DNA as set forth inany one of claims 35, 36 and 37, and an expression vector containing DNAas set forth in any one of claims 38, 39 and 40, and recovering thetarget antibody.
 75. A process for producing chimeric antibody againsthuman IL-8 comprising the steps of culturing host cells transformed withan expression vector containing DNA as set forth in any one of claims35, 36 and 37, and DNA as set forth in any one of claims 38, 39 and 40,and recovering the target antibody.
 76. A process for producing reshapedhuman antibody against human IL-8 comprising the steps of culturing hostcells transformed simaltaneously with an expression vector containingDNA as set forth in any one of claims 63, 64, 65 and 66, and anexpression vector containing DNA as set forth in any one of claims 67,68, 69, 70 and 71, and recovering the target antibody.
 77. A process forproducing reshaped human antibody against human IL-8 comprising thesteps of culturing host cells transformed with an expression vectorcontaining DNA as set forth in any one of claims 63, 64, 65 and 66, andDNA as set forth in any one of claims 67, 68, 69, 70 and 71, andrecovering the target antibody.